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.     

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.     

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.     

Distribution of heme oxygenase-2 and NADPH-diaphorase in the spinal trigeminal nucleus of the rat

Heme oxygenase (HO)/carbon monoxide (CO) and nitric oxide synthase (NOS)/nitric oxide (NO) systems are involved in sensory information processing. The present study was undertaken to examine the distribution of HO-2 and NOS in the spinal trigeminal nucleus (STN) of the rat, using histochemistry and immunohistochemistry. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining was found that NADPH-d activity was more prominent in the nucleus caudalis (Vc) and the dorsomedial subdivision of the nucleus oralis (Vo) than in other spinal trigeminal regions. Immunohistochemistry for HO-2 revealed that HO-2 staining neurons distributed extensively, which intensity was higher in the rostral than caudal part of the STN. The colocalization of NADPH-d and HO-2 was mainly confined in the Vc. The expression and distribution of NADPH-d and HO-2 suggest that NO and CO are likely neurotransmitters and might function in the processing orofacial signal in the STN together.     

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.     

NADPH-diaphorase and nitric oxide synthase in the canine superior cervical ganglion

By means of NADPH-diaphorase (NADPH-d) histochemistry and nitric oxide synthase (NOS) immunohistochemistry, we demonstrate that considerable numbers of NADPH-d-positive neurons are distributed throughout the canine superior cervical ganglion (SCG). These neurons also show NOS immunoreactivity. This finding indicates that NADPH-d histochemistry, a simple and reliable technique, can be used as a reliable marker of NOS activity in the sympathetic innervation of canine head and neck. The present findings suggest that the participation of nitric oxide in the SCG differs greatly between species.     

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.     

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.     

Prenatal hypoxic-ischemic insult changes the distribution and number of NADPH-diaphorase cells in the cerebellum

Astrogliosis, oligodendroglial death and motor deficits have been observed in the offspring of female rats that had their uterine arteries clamped at the 18(th) gestational day. Since nitric oxide has important roles in several inflammatory and developmental events, here we evaluated NADPH-diaphorase (NADPH-d) distribution in the cerebellum of rats submitted to this hypoxia-ischemia (HI) model. At postnatal (P) day 9, Purkinje cells of SHAM and non-manipulated (NM) animals showed NADPH-d+ labeling both in the cell body and dendritic arborization in folia 1 to 8, while HI animals presented a weaker labeling in both cellular structures. NADPH-d+ labeling in the molecular (ML), and in both the external and internal granular layer, was unaffected by HI at this age. At P23, labeling in Purkinje cells was absent in all three groups. Ectopic NADPH-d+ cells in the ML of folia 1 to 4 and folium 10 were present exclusively in HI animals. This labeling pattern was maintained up to P90 in folium 10. In the cerebellar white matter (WM), at P9 and P23, microglial (ED1+) NADPH-d+ cells, were observed in all groups. At P23, only HI animals presented NADPH-d labeling in the cell body and processes of reactive astrocytes (GFAP+). At P9 and P23, the number of NADPH-d+ cells in the WM was higher in HI animals than in SHAM and NM ones. At P45 and at P90 no NADPH-d+ cells were observed in the WM of the three groups. Our results indicate that HI insults lead to long-lasting alterations in nitric oxide synthase expression in the cerebellum. Such alterations in cerebellar differentiation might explain, at least in part, the motor deficits that are commonly observed in this model.     

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.     

NADPH-diaphorase expression in the rat jejunum after intestinal ischemia/reperfusion

The purpose of this study was to analyze the nicotinamide adenine dinucleotide phosphate - diaphorase (NADPH-d) activity in the rat jejunum after a mesenteric ischemia/reperfusion injury. Nitric oxide, synthetised from L-arginine by the enzyme nitric oxide synthase, is a nonadrenergic noncholinergic relaxant neurotransmitter of the intestinal smooth muscle. It plays an important role in the process of plasticity after the ischemia/reperfusion injury. Experimental animals were divided in two groups: the control group and the ischemic/reperfusion group, with different period of the reperfusion. The NADPH-d histochemical method has been used as a marker for the nitric oxide synthase. NADPH-d activity has been rapidly decreased in the neurons of both enteric nervous systems in plexuses of the jejunum after 1 h mesenteric ischemia and 1 h reperfusion. Differences were predominantly detected in the myenteric plexus; they were seen in change of the neuronal shape, in the arrangement of neurons and in intensity of their staining. The NADPH-d positivity was absent in the intestinal crypts. After 1 h ischemia and 24 h reperfusion, the NADPH-d activity was gradually increased, but it was lower in comparison with the control group. On the 30th day following the ischemia/reperfusion there were no changes in NADPH-d positivity compared with the control animals. These results indicated that the jejunal ischemia/reperfusion has affected the neurons of the enteric nervous system of adult rats and resulted in the early decrease of NADPH-d positivity 1 h of the reperfusion insult. The gradual increasing of NADPH-d activity in 24 h following the reperfusion could be considered as a result of the plasticity process. On the 30(th) day after the ischemia/reperfusion all histochemical changes were returned to the control levels.     

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.     

Ontogeny of NADPH-d expression in the thymic microenvironment of the chick embryo

Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry was used to demonstrate the presence of nitric oxide in the developing chicken thymus. NADPH-d was first expressed in the epithelial cells located at the corticomedullary junction of the thymic rudiment on day 13 of incubation. The number of labelled cells gradually increased from day 13 to day 21. Ultrastructural evidence showed that the labelling was localized in a heterogeneous population of cells in the medulla near the corticomedullary junction, comprising the cystic, undifferentiated, myoid, lymphoid and epithelial reticular cells. At this age, the vascular endothelium was NADPH-d positive. Labelling was also detected in some macrophages. The reaction product primarily labelled profiles of rough endoplasmic reticulum and to a lesser extent the outer membranes of mitochondria, portions of the nuclear envelope and the Golgi apparatus. By day 18/19, NADPH-d-labelled nerve fibres were occasionally observed in the interlobular connective tissue. By day 21, these fibres formed perivascular plexuses. Labelled nerve fibres were occasionally observed in the medullary parenchyma. Possible functions of nitric oxide in the embryonic thymus are discussed.     

An experimental, NADPH-diaphorase histochemical and immunocytochemical study of Mesocestoides vogae tetrathyridia.

In order to test the role of nitric oxide in flatworms, Mesocestoides vogae tetrathyridia were incubated together with L-arginine, which is the substrate for nitric oxide synthesis, or with NG-nitro-L-arginine, which is an irreversible inhibitor of nitric oxide synthase. Normally, tetrathyridia attach to each other with the aid of their suckers, forming clusters. The rate of cluster formation was followed during the incubations. L-Arginine stimulated, and NG-nitro-L-arginine clearly inhibited, the cluster formation. This is the first time that an effect of nitric oxide has been observed in a flatworm. In addition, the pattern of the NADPH-diaphorase histochemical reaction in the nervous system and the pattern of F-actin filaments in the musculature stained with TRITC-labelled phalloidin were studied. NADPH-d staining occurred in the brain and the main nerve cords but also followed the muscle fibres stained with phalloidin. The pattern of the NADPH-d reaction was compared with that of 5-HT immunoreactivity. The implications of the results are discussed in relation to the background of data on neuronal signal substances in M. vogae.     

NADPH-Diaphorase Activity in Normally Developing and Intracranially Transplanted Retinas

The activity and distribution of nicotinamide dinucleotide phosphate diaphorase (NADPH-d), an enzyme that is widely distributed in the central nervous system and involved in the production of the free radical nitric oxide, were investigated histochemically in the normal developing and intracranially transplanted retinas. In the normal rat retina, NADPH-d activity was first detected in cells in the ganglion cells layer (GCL) and blood vessels on the first postnatal day (P0). A small but distinct population of NADPH-d positive cells were observed along the inner border of the inner nuclear layer at P7. NADPH-d positive sublaminae began to appear in the inner plexiform layer during the second postnatal week, and several strongly reactive sublaminae resembling those observed in the adult were observed by the fourth postnatal week. The overall spatio- temporal sequence of development of NADPH-d positive cells in the transplanted retina was similar to that of the normal retina, except a lack of reactive in the inner plexiform layer in more mature transplants as compared with normal retinas of corresponding ages. These results indicate that the time course of development and distribution of NADPH-d cells in early postnatal retina requires signals mainly of intraretinal origin and is independent of influence from the surroundings. While this finding is supportive to the notion that neurons that are rich in NADPH-d are resistant to injury or perturbation, the observation of a lack of well organized NADPH-d reactive sublaminae in the inner plexiform layer in older transplants suggests a possible alteration in the synaptic circuitry in the inner retina with increasing postgrafting survival time.     

Comparison of NADPH diaphorase activity in the brains of hamsters infected with scrapie strains 139H or 263K or with normal hamster brain homogenate

Previous studies showed that the histopathological changes found in the brains of scrapie-infected animals included amyloid plaque formation, vacuolation, gliosis and neuronal and neurite degeneration. There were differences in the histopathological findings as a function of the scrapie strain-host combination. NADPH-diaphorase (NADPH-d) has been shown to be a selective histochemical marker for neurons containing nitric oxide (NO) synthase. Neuronal cell damage caused by NOS in brain has been reported to be associated with many neurodegenerative diseases. In this study, we used NADPH-d histostaining to investigate changes in the NOS system in brains of 139H- and 263K-infected hamsters and compared the results to normal hamster brain (NHB) injected animals. We observed that some of the NADPH-d histostaining neurons in the cortex of scrapie-infected hamsters appeared to be atrophic: the neurons were smaller and had fewer neurites. The NADPH-d histostaining intensity of neurons or astrocytes in septum, thalamus, hypothalamus and amygdala of 139H- and 263K-infected hamsters was greater than in control hamsters. Astrocytes in the thalamus, hypothalamus and lower part of the cortex (layers 4 to 6) in 263K-infected hamsters were more intensely stained for NADPH-d than in either 139H-infected hamsters or controls. Our results suggest that changes in NADPH-d system might play a role in the diversity of scrapie induced neurodegenerative changes.     

Differential histochemical localization patterns of reduced nicotinamide adenine dinucleotide phosphate-diaphorase and neuronal nitric oxide synthase during postnatal development of the rat vomeronasal organ

The present study was undertaken to examine the localization patterns of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) by enzyme histochemistry and neuronal nitric oxide synthase (NOS) by immunohistochemistry in the vomeronasal organ of rat from postnatal day 0 for 8 weeks (adult). Nicotinamide adenine dinucleotide phosphate-diaphorase activity was not observed in the sensory epithelium of the vomeronasal organ at postnatal day 0 (the day of birth) and at day 1. At postnatal day 2, NADPH-d activity was observed in several vomeronasal neurons and on the surface of the sensory epithelium. From 25 days through adulthood, the number of vomeronasal neurons having NADPH-d activity increased gradually. On the other hand, neuronal NOS immunoreactivity was not observed in the sensory epithelium of the vomeronasal organ in newborns or in the adult rat. In this study, it is suggested that the nitric oxide pathway in the sensory epithelium of the vomeronasal organ comes into play beyond postnatal day 3. Moreover, it was found that NADPH-d and neuronal NOS are not colocalized in the sensory epithelium of the developing rat vomeronasal organ.     

Histochemical and immunohistochemical localization of neuronal nitric oxide synthase in the olfactory epithelium of the axolotl, Ambystoma mexicanum.

The aim of this study was to describe the anatomic distribution of neuronal nitric oxide synthase immunoreactivity (nNOS-IR) and nicotinamide-adenine dinucleotide phosphate-diaphorase (NADPH-d) staining in the olfactory epithelium of the axolotl, juvenile, and neotenic adult, Ambystoma mexicanum. Nitric oxide (NO, nitrogen monoxide) is a widespread molecule that has been identified both as a neuromodulator and as an intracellular messenger. In the olfactory system, NO has been proposed to play a role in olfactory transduction. Nitric oxide synthase (NOS) can be detected by histochemical (NADPH-d) and immunohistochemical techniques. NADPH-d staining has been described in olfactory receptor neurons (ORN) of several species; however, nNOS-IR has not always been found at ORN. Present results show intense NADPH-d staining and nNOS-IR in the dendrites and cell bodies of ORN in both the nasal cavity and the vomeronasal organ of axolotls. Unilateral olfactory axotomy was conducted to confirm that labels were at ORN. Two weeks after this procedure an important decrease in NADPH-d staining and nNOS-IR was observed. The remaining labels were mostly in basal cells. By 5 weeks postaxotomy both labels were almost totally absent. Thus, both NADPH-d staining and nNOS-IR were mainly localized in ORN. NADPH-d staining and nNOS-IR were also found in nerve fibers surrounding arterioles, as well as in secretory and duct cells of the Bowman's glands. This last anatomical localization suggests that in the A. mexicanum NO might be involved in functions other than only olfactory transduction, such as regulation of local blood flow, glandular secretion, and ORN development.     

Effects of orlistat and its relationship with nitric oxide in the small intestinal mucosa

Nitric oxide (NO) is known to be a messenger molecule that plays an important role in physiological and pathological conditions. It is synthesized by an enzyme called nitric oxide synthase (NOS). Inducible NOS (iNOS), one of the three isomers of NOS, has both protective and toxic properties. In this study, the role of NO has been evaluated by gastrointestinal symptoms induced by orlistat which is used in obesity treatment. Orlistat was given to Wistar rats with and without iNOS inhibition. The effects of orlistat and inhibition of NOS were studied. Glucose, urea, alanine transaminase (ALT), and gamma glutamil transpeptidase (GGT) were descreased after short- and long- term orlistat applications. Dexamethasone increased level of these enzymes. Cholesterol and triglyceride were increased in all experimental groups than the controls. This increment was more severe in animals received orlistat and dexamethasone together. Small intestinal tissue also were researched histologically and NADPH-diaphorase (NADPH-d) histochemistrically. Orlistat caused histological damages in brush border membranes, connective tissues of villi, and lymphocyte migration also increased. Dexamethasone treatment prevented these damages partially while orlistat increased the NOS distribution in the tissue sections. Dexamethasone, which is an iNOS inhibitor, decreased NADPH-d histochemistry. There was a similiar NOS distribution both in the control and orlistat+dexamethasone group. Hence, we concluded that long- term trials with orlistat and similar drugs are needed.     

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.