Ultrastructural localization of NADPH-diaphorase and colocalization of nitric oxide synthase in endothelial cells of the rabbit aorta

This is the first report on the ultrastructural pattern of distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in endothelial cells, using the rabbit aorta, and its colocalization with the neuronal isoform (type I) of nitric oxide synthase. About 30% of the endothelial cells showed a positive reaction for NADPH-d compared to about 6% for nitric oxide synthase immunoreactivity. Simultaneous double histochemical-immunocytochemical labelling procedures indicate that all of the cells displaying nitric oxide synthase-positive reactivity also contained NADPH-d; the remainder of NADPH-d-positive endothelial cells were negative for this isoform of nitric oxide synthase. Nitric oxide synthase-immunogold labelling was mostly associated with free ribosomes, while NADPH-d activity was distributed largely in patches in the cytoplasm and in association with the cell membrane.     

Neuronal and endothelial nitric oxide synthase immunoreactivity and NADPH-diaphorase staining in rat and human pancreas: influence of fixation

In this study, we wished to clarify the distribution and co-localization of nitric oxide synthase and NADPH-diaphorase (NADPH-d) in nerve cells, nerve fibres and parenchymal cells in exocrine and endocrine pancreas, and to assess the influence of fixation on the staining pattern obtained. For this purpose, we applied nitric oxide synthase immunocytochemistry and NADPH-d histochemistry to rat and human pancreas under different fixation conditions. Antibodies to neuronal and endothelial nitric oxide synthase were similarly applied. We found complete co-localization of neuronal nitric oxide synthase and NADPH-d in ganglion cells, and in nerve fibres around acini, excretory ducts, blood vessels and in islets of Langerhans of rat and human pancreas. Immunoreactivity for endothelial nitric oxide synthase was co-localized with NADPH-d in endothelial cells. However, in NADPH-d reactive islet and ductal epithelial cells we could detect neither brain nor endothelial nitric oxide synthase immunoreactivity with any fixation protocol applied. There were marked differences in NADPH-d staining of both neurons and parenchymal cells under different fixation conditions. These results indicate the existence of different types of NADPH-d, which are associated or not associated with nitric oxide synthase(s), and which are differently influenced by various fixation procedures in rat and human pancreas.     

NADPH-diaphorase expression in the meibomian glands of rat palpebra in postnatal development

In the current study, we aimed at investigating the presence of nitric oxide synthase (NOS) positive nerve fibers in rat meibomian glands (MGs) at various stages of development. There is good evidence to suggest that nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) is a surrogate for neuronal nitric oxide synthase (NOS). Sections of the central, upper eyelids of Wistar rats were processed histochemically for NADPH-d to investigate the presence and distribution of NOS-positive nerve fibers at the following time points: day 1 and weeks 1, 2 and 3 post partum, and in adult controls. At day 1, MG acini were lightly stained and located at a distance from the mucosal border. Vessels were accompanied by intensely stained NADPH-d positive nerve fibers. At the week 1 time point, both the vessels and the NADPH-d positive fibers were still present, but less numerous. MGs were now closer to the mucosa, so that the submucosa was thinner. The acini were mostly pale but occasionally darker. At week 3, there were fewer blood vessels in both the submucosa and within the septa. Darker acini were more common than lightly stained acini. NADPH-d positive dots were observed in the vicinity of the MGs. At the week 3 time point, MGs were adjacent to the mucosal border and stained more intensely than at earlier times; almost all acini were stained. The microscopic appearances were almost identical with those of adult palpebra. Submucosal and septal blood vessels and NADPH-d positive nerve fibers were less numerous. NADPH-d histochemical staining confirmed differences in the density of stained nerve fibers at different developmental stages. The greatest density of NADPH-d -positive nerve fibers occurred in 1-day-old rats whereas they were less numerous in adult rat eyelids. Nerves innervating MGs utilize nitric oxide (NO) as a neurotransmitter mostly in early developmental stages and this need thereafter decreases and stabilizes at 3 weeks postnatally.     

Expression and colocalization of NADPH-diaphorase and heme oxygenase-2 in trigeminal ganglion and mesencephalic trigeminal nucleus of the rat

Carbon monoxide (CO) and nitric oxide (NO) are two endogenously produced gases that can function as second messenger molecules in the nervous system. The enzyme systems responsible for CO and NO biosynthesis are heme oxygenase (HO) and nitric oxide synthase (NOS), respectively. The present study was undertaken to examine the distribution of HO-2 and NOS of the trigeminal primary afferent neurons of the rat, located in the trigeminal ganglion (TG) and mesencephalic trigeminal nucleus (MTN), using histochemistry and immunohistochemistry. NADPH-d staining was found in most neurons in TG. The intensely NADPH-d-stained neurons were small- or medium-sized, while the large-sized neurons were less intensely stained. Immunocytochemistry for HO-2 revealed that almost all neurons in TG expressed HO-2, but they did not appear cell size-specific pattern. NADPH-d and HO-2 positive neurons appeared the same pattern, which was NADPH-d activity and HO-2 expression progressively declined from the caudal to rostral part of the MTN. A double staining revealed that the colocalization of NADPH-d/HO-2 neurons was 97.3% in TG and 97.6% in MTN. The remarkable parallels between NADPH-d and HO-2 suggest that NO and CO are likely neurotransmitters and mediate the orofacial nociception and sensory feedback of the masticatory reflex arc together.     

Distribution of NADPH-diaphorase and nitric oxide synthase in the trigeminal ganglion and mesencephalic trigeminal nucleus of the cat. A histochemical and immunohistochemical study

The trigeminal ganglion (TrG) and mesencephalic trigeminal nucleus (MTN) neurons are involved in the transmission of orofacial sensory information. The presence of nitric oxide (NO), a putative neurotransmitter substance in the nervous system, was examined in the cat TrG and MTN using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry. In the TrG, where the majority of the trigeminal primary afferent perikarya are located, most of the intensely NADPH-d/ NOS-stained cells were small in size and distributed randomly throughout the ganglion. The medium-sized neurons were moderately stained. A plexus of pericellular varicose arborizations around large unstained ganglion cells and densely stained fibers in-between could also be observed. In the caudal part of the MTN, both NADPH-d activity and NOS immunoreactivity was present in MTN neurons. In addition, a few scattered NADPH-d/NOS-containing neurons were found in the mesencephalic-pontine junction part of the nucleus. In contrast, only nerve fibers and their terminals were present at a more rostral level in the mid- and rostral MTN. MTN neuronal perikarya were enveloped in fine basket-like NADPH-d/ NOS-positive networks. Differential expression patterns of NOS and its marker NADPH-d suggest that trigeminal sensory information processing in the cat MTN is controlled by nitrergic input through different mechanisms. We introduce the concept that NO can act as a neurotransmitter in mediating nociceptive and proprioceptive information from periodontal mechanoreceptors but may also participate in modulating the activity of jaw-closing muscle afferent MTN neurons.     

Neuronal nitric oxide synthase in the rabbit spinal cord visualised by histochemical NADPH-diaphorase and immunohistochemical NOS methods

The NADPH-diaphorase (NADPH-d) staining method is widely used in the investigation of both the central and peripheral nervous systems. Neuronal nitric oxide synthase (nNOS) has previously been shown to be responsible for the NADPH-d activity in neurons. However, NADPH-d activity does not always fully represent the enzyme nNOS. We investigated the distribution of NADPH-d activity and nNOS protein in the rabbit spinal cord for all groups of neurons and Rexed's laminae. In most laminae the distribution of NADPH-d activity was identical to nNOS immunoreactivity. Both were present in the dorsal horn and in pericentral areas of the spinal cord, but some differences existed. The superficial part of the dorsal horn (laminae I-III) stained more intensely for NADPH-d than for nNOS. However, the most prominent difference was seen in the lateral part of the dorsal horn--the lateral collateral pathway (LCP). The LCP stained strongly for NADPH-d activity, while nNOS staining was absent. Although there is an excellent correlation between NADPH-d staining and nNOS immunohistochemical staining in the spinal cord in general, the presence of staining differences necessitates the use of immunohistochemistry for some specialized applications.     

Localization of nitric oxide synthase in rat trigeminal primary afferent neurons using NADPH-diaphorase histochemistry

Summary Nitric oxide (NO) is a ubiquitous gaseous neurotransmitter that has been ascribed to a large number of physiological roles in sensory neurons. It is produced by the enzyme nitric oxide synthase (NOS). To identify the NOS-containing structures of rat trigeminal primary afferent neurons, located in the trigeminal ganglion (TrG) and mesencephalic trigeminal nucleus (MTN), histochemistry to its selective marker nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) was applied in this study. In the TrG approximately half of the neuronal population was NADPH-d reactive. Strongly positive were neurons mainly of small-to-medium size. Neuronal profiles of large diameter were less intensely stained. In addition, NADPH-d-positive nerve fibers were dispersed throughout the ganglion. Nitrergic neurons were located in the caudal part and mesencephalic-pontine junction of the MTN. Most of them were large-sized pseudounipolar cells. In a more rostral aspect, the reactive psedounipolar MTN profiles gradually decreased in number and intensity of staining. There, only a fine meshwork of stained thin fibers and perisomatic terminal arborizations, and also some isolated perikarya of NADPH-d stained multipolar MTN neurons, were observed. The predominant NADPH-d localization in smaller in size TrG neurons, which are considered nociceptive, suggests that NO may play a role in the pain transmission in the rat trigeminal afferent pathways. In addition, the wide distribution of NADPH-d activity in large pseudounipolar and certain multipolar MTN neurons provides substantial evidence that NO may also participate in mediating proprioceptive information from the orofacial region. The differential expression patterns of nitrergic fibers in the TrG and MTN suggest that trigeminal sensory information processing is controlled by nitrergic input through different mechanisms.     

NADPH-diaphorase activity in the nervous system of the embryonic and juvenile pond snail, Lymnaea stagnalis

Nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d) histochemistry has been applied in the present study to determine the distribution of putative nitric oxide (nitric oxide synthase)-producing cells during embryonic and early postembryonic development in the pond snail, Lymnaea stagnalis L., with special reference to the nervous system. The first NADPH-d-positive structures appear as early as 18% of development (E18, trochophore stage) and correspond to the pair of protonephridia. These structures later show disintegration, although after metamorphosis (E26=75%) staining of their individually spreading cells can be observed until hatching. Peripheral sensory neurons in the foot, mantle edge and lips, and their afferents projecting to the central nervous system reveal NADPH-d activity in the postmetamorphosis period (E25–E27=E60%–E80%) of embryogenesis. After hatching (P1–P3), a number of stained sensory cells appear in the pharynx and esophagus. Some NADPH-d positive neuronal perikarya occur in the pedal and pleural ganglia, and a few weakly stained cells in the cerebral and buccal ganglia of juvenile snails. At the same time, a continuous bundle of reactive fibers is formed in the neuropil both through and through around the circumesophageal ganglion ring. The localization of NADPH-d activity in the developing nervous system of Lymnaea suggests that nitric oxide participates mainly in sensory processes. However, its role in specific intraganglionic integrative events cannot be excluded following embryonic metamorphosis.     

Histochemical localization of NADPH-d-positive structures in bovine uterine and ovarian arteries during the oestrous cycle and early pregnancy

The aim of the present study was to histochemically demonstrate nitric oxide synthase-related NADPH-d activity in ovarian and uterine arteries of heifers at different stages of the oestrous cycle and during early pregnancy. Catalytic activity of NADPH-d activity was found in the endothelial lining of all examined vessels, however, staining intensity was higher in the segments ipsilateral to the corpus luteum than in those taken from the contralateral side. Moreover, the reaction was much more intense during the luteal than during the follicular stage of the cycle. Similar differences were observed for NADPH-d activity in the muscular coat. In conclusion, the present results suggest that the endothelial/muscular cells may be the main source of nitric oxide in the studied parts of the bovine arteries, and also that NADPH-d activity may depend on the hormonal status of the organism.     

Enzyme-cytochemically detectable NADPH-diaphorase activity is present in the endoplasmic reticulum and nuclear envelope of the syncytiotrophoblast of the human placenta

We investigated the subcellular localization of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity, a histo- and cyto-chemical marker of nitric oxide synthase, in human placental trophoblast obtained from women with normal term pregnancies. Tetrazolium salt BSPT was used as the capturing agent. Precipitates of BSPT-formazan indicative of NADPH-d reaction were observed on the membranes of endoplasmic reticulum and nuclear envelope of syncytiotrophoblasts. Our results indicate these two intracytoplasmic organellae are the sites of nitric oxide generation in the syncytiotrophoblasts of normal term human placenta.     

Expression of peptidergic and nitrergic structures in dorsal root ganglia of the rabbit

In this study we have demonstrated the presence of neuropeptide substance P (SP)and nonpeptide neurotransmiter NO (nitric oxide) in the dorsal root ganglia (DRG) of rabbits. NADPH-diaphorase histochemical staining was used for detection of NO and an immunohistochemical method for detection of substance P. A number of DRG cells were stained by SP- and NADPH-d reactions. The presence of SP and NADPH-diaphorase positive cells varied depending upon the spinal level of the DRG. Positively stained neurons were only small and intermediate in size. Cells of large diameter profiles showed no staining. Substance P immunoreactive cells were of brown and dark brown colour, the intensity of NADPH-d staining varied from light to very dark blue. In some DRG cells, there was very significant neuronal co-localization of immunoreactivity for SP and reactivity for NADPH-d. In summary, DRG cells appear to express diaphorase and substance P activity, and some of them show the presence of both neurotransmitters. Recent studies on the participation of NO in the regulation of SP release in the spinal cord suggest, that also in the DRG neurons there may be a close interaction between NO and SP.     

Partial colocalization of NADPH-diaphorase and acetylcholinesterase positivity in spinal cord neurons

The freely diffusible radical, nitric oxide (NO), has been assumed to act as a retrograde signaling molecule that modulates transmitter release. Acetylcholine (ACh) is known to function as a typical neurotransmitter. In the present work we have examined the presence of both transmitters (NO and ACh) and their possible relations in the rabbit spinal cord. In our experiments we have used histochemical methods for the visualization of acetylcholinesterase (AChE) and NADPH diaphorase (NADPH-d) which label neurons that express nitric oxide synthase (NOS). Both histochemical methods were performed separately or together on the same sections of the thoracic spinal cord. NADPH-d positive dark blue stained neurons were seen mostly in superficial and deep layers of the dorsal horn, preganglionic autonomic neurons and pericentral area. The presence of AChE positive amber yellow neurons was confirmed mostly in motoneurons located in the ventral horns and in neurons of the pericentral and intermediate zone. Besides the above mentioned neurons, also double-labeled neurons were found which contained both the yellow and dark blue histochemical product. Their presence was confirmed in the intermediate zone and in the pericentral area. Thus, the co-existence of NADPH-d and AChE occurred in the location of interneurons. Our observations suggest that NO may play a role in the control of cholinergic neuronal activity and that NO can be involved in the modulation of synaptic transmission.     

Expression of nitric oxide synthase in mucosal cells of the canine colon

The expression of nitric oxide synthase (NOS) in the mucosa of the canine colon was investigated with in situ hybridzation, immunohistochemistry (using isoform specific antibodies), western analysis, and NADPH diaphorase (NADPH-d) histochemistry. In situ hybridization using a common probe for known isoforms of NOS showed that NOS mRNA was strongly expressed in mucosal cells. A gradient in the degree of hybridization was noted from the base of the crypts to the luminal surface. This gradient was also apparent using an endothelial NOS (eNOS)-specific probe. Neural NOS-like immunoreactivity (nNOS-LI) was observed in columnar epithelial cells, and the same population of cells was stained with NADPH-d. Endothelial NOS-like immunoreactivity (eNOS-LI) was also found in mucosal cells; however, this eNOS-LI was confined to mucous cells. These cells were not stained with NADPH-d. The existence of cNOS in mucosal cells was confirmed by in situ hybridization using the probe which specifically hybridized with mRNA of eNOS and by western blots which demonstrated the expression of a 135-kDa protein in mucosal homogenates. The differential expression of NOS isoforms and the gradient in expression along the length of the crypts suggest complex roles for NO in the development of colonic epithelial cells and in secretion and transport functions of the colonic mucosa.     

Localization of nitric oxide synthase in canine ileocolonic and pyloric sphincters

The distribution of neurons containing NADPH-diaphorase (NADPH-d) activity and nitric oxide synthase-like immunoreactivity (NOS-LI) in the canine pyloric and ileocolonic sphincters was studied. Cells within the myenteric and submucosal ganglia were positive for NADPH-d. These cells generally had the morphology of Dogiel type-I enteric neurons, however, there was some diversity in the morphology of NADPH-d-positive neurons in the myenteric plexus of the pylorus. Intramuscular ganglia were observed in both sphincters, and NADPH-d was found in a sub-population of neurons within these ganglia. Dual staining with an antiserum raised against nitric oxide synthase (NOS) demonstrated that almost all cells with NOS-LI were also NADPH-d positive. Varicose fibers within ganglia and within the circular and longitudinal muscle layers also possed NOS-LI and NADPH-d activity. Dual staining with anti-VIP antibodies showed that some of the NADPH-d-positive cells in the myenteric and submucosal ganglia also contained VIP-LI, but all VIP-LI-positive cells did not express NADPH-d activity. These data are consistent with recent physiological studies suggesting that nitric oxide serves as an inhibitory neurotransmitter in the pyloric and ileocolonic sphincters. The data also suggest that VIP is expressed in a sub-population of NADPH-d-positive neurons and may therefore act as a co-transmitter in enteric inhibitory neurotransmission to these specialized muscular regions.     

Detection of NADPH-diaphorase activity in Paramecium primaurelia

Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the cholinergic system and involved in modulating cell-cell interactions leading to the sexual process of conjugation. It is known that nitric oxide (NO) plays a role in regulating the release of transmitter molecules, such as acetylcholine, and that the NO biosynthetic enzyme, nitric oxide synthase (NOS), shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity. In this work, we detected the presence of NADPH-d activity in P. primaurelia. We characterized this activity histochemically by examining its specificity for beta-NADPH and alpha-NADH co-substrates, and sensitivity both to variations in chemico-physical parameters and to inhibitors of enzymes showing NADPH-d activity. Molecules immunologically related to NOS were recognized by the anti-rat brain NOS (bNOS) antibody. Moreover, bNOS immunoreactivity and NADPH-d activity sites were found to be co-localized. The non-denaturing electrophoresis, followed by exposure to beta-NADPH or alpha-NADH co-substrates, revealed the presence of a band of apparent molecular mass of about 124 kDa or a band of apparent molecular mass of about 175 kDa, respectively. In immunoblot experiments, the bNOS antibody recognized a single band of apparent molecular mass of about 123 kDa.     

Age-Related Changes of NADPH-Diaphorase Positivity in the Rat Rostral Migratory Stream

Summary Accumulating evidence confirms that nitric oxide (NO), a versatile diffusible signaling molecule, contributes to controling of adult neurogenesis. We have previously shown the timing of NADPH-diaphorase (NADPH-d) positivity within the rat rostral migratory stream (RMS) during the first postnatal month. The present study was designed to describe further age-related changes of NO presence in this neurogenic region. The presence of NO synthesizing cells in the RMS was shown by NADPH-d histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. The phenotypic identity of nitrergic cells was examined by double labeling with GFAP and NeuN. Systematic qualitative and quantitative analysis of NADPH-d-positive cells was performed in the neonatal (P14), adult(5 months) and aging (20 months) rat RMS. 1. Nitrergic cells with different distribution pattern and morphological characteristics were present in the RMS at all ages examined. In neonatal animals, small, moderately stained NADPH-d-positive cells were identified in the RMS vertical arm and in the RMS elbow. In adult and aging rats a few labeled cells could be also detected in the RMS horizontal arm. NADPH-d-positive cells in adult and aging rats were characterized by long varicose processes and displayed dark labeling in comparison to the neonatal group. 2. Double immunolabeling has revealed that nNOS-immunoreactivity co-localized with that of NeuN. This indicates that nitrergic cells within the RMS are neurons. 3. Quantitative analysis showed that the number of NADPH-d-positive cells increases with advancing age. The presence of NO producing cells in the RMS of neonatal adult and aging rats indicates, that this proliferating and migratory area is under the influence of NO throughout the entire life of the animals.     

Involvement of nitric oxide in inflammation of ovaries in gilts

NADPH-diaphorase (NADPH-d) and an inducible type of nitric oxide synthase (iNOS) were demonstrated in porcine ovaries after unilateral infusion of bacteria into the hilus of an ovary. In group I one ml of saline was infused into the hilus of each ovary from the 15th day to the 19th day of the estrous cycle. In group II one ml of bacterial suspension (10(9) colony forming units of Escherichia coli, Staphylococcus aureus and Corynebacterium pyogenes, in a proportion 1:1:1, respectively) in saline was infused into the hilus of one ovary on days corresponding to those of the control group (gr. I), whereas saline was infused into the contralateral ovary. The ovaries were collected on the 7th day of the next estrous cycle. In the bacteria-treated ovary, the activity of NADPH-d was higher in the endothelium of blood vessels, corpora lutea and follicular walls in comparison to that observed in the respective structures of the contralateral ovary. The highest activity of NADPH-d was found in the vascular endothelium in the bacteria-infused ovary. Vascular smooth muscle cells found in both ovaries of the bacteria-treated gilts were more intensely stained for NADPH-d than those in control animals. After bacteria administration, the intensity of NADPH-d reaction in all the structures of both ovaries in group II was higher than in control group. The strongest immunostaining for iNOS was observed in all structures of the bacteria-infused ovary. In the contralateral ovary, iNOS-immunoreactivity was weaker but still stronger than that in control group. The present results revealed that infusions of bacteria into the hilus of one ovary enhanced the activity of NADPH-d and immunoreactivity for iNOS in both porcine ovaries. However, the activity of both enzymes was higher in the bacteria-infused ovary than in the contralateral one. These data suggest that locally synthesized NO can mediate an inflammatory effect of bacteria in the porcine ovaries.     

Morphological and biochemical investigation of nitric oxide synthase and related enzymes in the rat and pig urothelium.

We investigated the enzymes involved in the NADPH-diaphorase (d) reaction in the rat and pig bladder urothelium. The urothelial cell layer displayed intense and uniform NADPH-d activity. Preincubation with the flavoprotein inhibitor diphenyleneiodionium chloride (DPI) and the alkaline phosphatase inhibitor levamisole concentration-dependently decreased the urothelial NADPH-d activity. Immunoreactivities to neuronal (n), endothelial (e), or inducible (i) nitric oxide synthase (NOS) were not detected in rat or pig urothelial cells. In rats, the urothelium was uniformly immunoreactive for NADPH cytochrome P450 reductase, whereas the pig urothelium displayed inconsistent labeling. In lipopolysaccharide (LPS)-treated rats, the bladder urothelium showed positive iNOS immunoreactivity. The iNOS labeling was found predominantly in cells located in the basal layer of the urothelium. In the pig bladder mucosa, a Ca2+-dependent NOS activity was evident in cytosolic and particulate fractions that was quantitatively comparable to the NOS activity found in the smooth muscle. In ultrastructural studies of urothelial cells, NADPH-d reaction products were found predominantly on membranes of the nuclear envelope, endoplasmatic reticulum and mitochondria. In conclusion, NADPH-d staining of the urothelium cannot be taken as an indicator for the presence of constitutively expressed NOS. Activity of alkaline phosphatase and cytochrome P450 reductase may account for part of the NADPH-d reaction in urothelial cells. However, LPS treatment of rats caused expression of iNOS in urothelial cells.