Reaction of Mast Cells in the Zone of Polypropylene Mesh Implantation.

Reaction of mast cells of adult male Wistar rats (n=15) in the zone of polypropylene mesh fixation was studied by histochemical, immunohistochemical, and traditional morphological methods on days 1, 5, 10, and 30 after implantation. Immediately after the intervention, mast cells stimulated the processes aimed at wound healing. Secretion of mast cells was clearly regulatory. These cells migrated to the zone of injury for subsequent activation of their function. The number of cNOS+ mast cells near the polypropylene mesh was maximum on day 1 and the number of iNOS+ mast cells peaked on day 5 of the experiment, which probably represented a compensatory reaction. Presumably, stimulation of fibrillogenesis was largely due to the activatory effect of mast cells on the fibroblast function, but not to collagen production by these mast cells.

Old Yellow Enzyme from Trypanosoma cruzi Exhibits In Vivo Prostaglandin F2α Synthase Activity and Has a Key Role in Parasite Infection and Drug Susceptibility.

The discovery that trypanosomatids, unicellular organisms of the order Kinetoplastida, are capable of synthesizing prostaglandins raised questions about the role of these molecules during parasitic infections. Multiple studies indicate that prostaglandins could be related to the infection processes and pathogenesis in trypanosomatids. This work aimed to unveil the role of the prostaglandin F2α synthase TcOYE in the establishment of Trypanosoma cruzi infection, the causative agent of Chagas disease. This chronic disease affects several million people in Latin America causing high morbidity and mortality. Here, we propose a prokaryotic evolutionary origin for TcOYE, and then we used in vitro and in vivo experiments to show that T. cruzi prostaglandin F2α synthase plays an important role in modulating the infection process. TcOYE overexpressing parasites were less able to complete the infective cycle in cell culture infections and increased cardiac tissue parasitic load in infected mice. Additionally, parasites overexpressing the enzyme increased PGF2α synthesis from arachidonic acid. Finally, an increase in benznidazole and nifurtimox susceptibility in TcOYE overexpressing parasites showed its participation in activating the currently anti-chagasic drugs, which added to its observed ability to confer resistance to hydrogen peroxide, highlights the relevance of this enzyme in multiple events including host-parasite interaction.

Betulinic acid­induced expression of nicotinamide adenine dinucleotide phosphate­diaphorase in the immune organs of mice: A possible role of nitric oxide in immunomodulation.

The aim of the present study was to investigate the effects of betulinic acid (BetA) on the expression and distribution pattern of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH­d), an indirect indicator of nitric oxide (NO) synthase in the thymus and spleen of mice. Mice were randomly assigned to four main groups (n=48 per group): Experimental group (BetA), positive control group (goniothalamin), vehicle control group (dimethyl sulfoxide) and control group (without vehicle). Each group was further divided into three equal subgroups according to the treatment length (4, 8 and 12 days). BetA treatment induced the expression of NADPH­d activity in the thymus and spleen without any significant changes in the morphology of the organs. Furthermore, the expression pattern of NADPH­d in BetA­treated animals was significantly increased compared with that in the control animals. NADPH­d expression in the thymus and spleen suggests that NO signaling may be a potential mechanism underlying the BetA­induced immunomodulation in these organs. These findings are of direct clinical relevance and may contribute to the further development of BetA as a therapeutic drug.

The spatial relationship between the musculature and the NADPH-diaphorase activity, 5-HT and FMRFamide immunoreactivities in redia, cercaria and adult Echinoparyphium aconiatum (Digenea).

The spatial relationship between the musculature and the NADPH-diaphorase (NADPH-d) activity, 5-HT and FMRFamide immunoreactivities in redia, cercaria and adult Echinoparyphium aconiatum was studied using scanning electron microscopy (SEM), NADPH-d histochemistry, immunocytochemistry, and confocal scanning laser microscopy (CSLM). TRITC-conjugated phalloidin was used to stain the musculature. Staining for NADPH-d was observed in the central (CNS) and peripheral nervous system (PNS) of all three stages. NADPH-d positive nerves occurred very close to muscle fibres. 5-HT-immunoreactive (5-HT-IR) nerve cells and fibres occurred in the CNS and PNS and close to muscle fibres. FMRFamide-IR nerve fibres were observed in the CNS and PNS of adult worms. This is the first time, the presence of the NADPH-d has been demonstrated in the larval as well as the adult stages of a fluke.

Distribution of constitutive nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in murine telogen and anagen skin.

The freely diffusible radical nitric oxide is generated by nitric oxide synthase, and is a pleiotropic, bioregulatory molecule that regulates, e.g., the vascular tone, functions as a major neurotransmitter, and is involved in macrophage-mediated cytotoxicity and platelet aggregation. Constitutive nitric oxide synthase exhibits NADPH-diaphorase activity that can be demonstrated histochemically. To study whether this enzyme is present in mammalian skin during distinct phases of the murine hair cycle, we have examined cryosections of C 57 BL-6 mouse skin in telogen and depilation-induced anagen VI. Histochemical analysis of NADPH-diaphorase activity was complemented by immunohistology, using two specific rabbit antisera against constitutive neuronal nitric oxide synthase. Epidermis and the outer root sheath showed both immunoreactivity for the enzyme and NADPH-diaphorase activity, whereas dermal papilla and sebaceous glands displayed only strong NADPH-diaphorase activity, suggesting that this enzyme histochemical test measures additional enzymes besides nitric oxide synthase. Intrinsic nitric oxide synthase immunoreactivity was also detected by immunoblot in mouse skin homogenates, staining proteins of an apparent 160-kDa molecular weight. Compared to telogen skin, these immunoreactive proteins were quantitatively increased in anagen VI skin. Thus, our study suggests that defined epithelial compartments of normal murine skin are capable of synthesizing nitric oxide and that the molecule may be involved in skin physiology, growth, and remodeling.

Analysis of cell death in the trochlear nucleus of the chick embryo: calibration of the optical disector counting method reveals systematic bias.

Detection of changes in numbers of neurons is essential for an understanding of neuronal development, function, and death. Optical disector counting is claimed to be the most efficient technique to estimate accurate numbers of neurons in microscopic sections. We calibrated the optical disector by comparison with three-dimensional reconstructions from serial sections and determined how accurate this technique is relative to conventional profile counting methods. The calibration was performed on the trochlear nucleus in developing chicks. Optical disector estimates, when obtained as generally recommended, were about 25% lower than the actual number of neurons. This underestimate was caused by a nonuniform (bimodal) distribution of neuronal nuclei in paraffin and plastic (glycolmethacrylate) sections, but not in cryosections. The density of neurons in the core of the paraffin and plastic sections was substantially lower than in the upper and lower margins of these sections. Accurate estimates of neuronal numbers were obtained with a modified optical disector method that sampled the entire extent of tissue sections. Previous estimates of numbers of trochlear neurons in the developing chick have been controversial. The modified (calibrated) optical disector method revealed that the number of trochlear neurons decreased from about 1,600 at day 8.5 of incubation (embryonic day, [E] 8.5) to about 900 at the time of hatching. Numbers of pyknotic nuclei peaked at E6 and at E9, revealing an additional early, but postproliferative, period of cell death. Taken together, these data emphasize the need for calibration of stereological counting techniques and the need to examine sampling strategies for potential bias.

Nitric oxide-containing neurons in the nervous ganglia of Helix aspersa during rest and activity: immunocytochemical and enzyme histochemical detection.

Nitric oxide synthase (NOS) immunoreactivity and staining for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-diaphorase) activity are two cytochemical markers for nitric oxide (NO)-containing neurons. The authors examined the changes in the distribution of NOS immunolabeling and NADPH-diaphorase reactivity in the cerebral and buccal ganglia of the terrestrial snail Helix aspersa during resting and active phases. During inactivity and after 1 day of activity, in the mesocerebrum and metacerebrum of the snails, there were several reactive neurons for both markers; after 7 days of activity, the number of reactive neurons was lower. Opposite results were obtained in the buccal ganglia, in which increased staining and numbers of reactive neurons were present in the active snails (after 1 day and 7 days of activity). Although the staining patterns for the two reactions were similar, colocalization was not always observed. The comparison between inactive and active animals provided a more precise survey of NOS-containing neurons in the snail cerebral ganglia than previously described. Moreover, it suggested that not only is NO involved in distinct nervous circuits, but, as a ubiquitous molecule, it also plays a role in neuroprotection and neuropeptide release.

Identifiable nitrergic neurons in the central nervous system of the nudibranch Melibe leonina localized with NADPH-diaphorase histochemistry and nitric oxide synthase immunoreactivity.

Nitric oxide (NO) is a gaseous intercellular messenger produced by the enzyme nitric oxide synthase (NOS). In this study, we used two different techniques-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and NOS immunocytochemistry-to demonstrate that NOS is present in a pair of identifiable cells in the central nervous system of the nudibranch Melibe leonina. In the Melibe brain, NADPH-d histochemistry revealed only a single pair of bilaterally symmetrical cells in the cerebropleural ganglia. NOS activity also was found in the neuropil of the cerebral, pedal, and buccal ganglia; in the tentacles of the oral hood; in the sensory end of the rhinophores; and in the epithelial tissue of the mouth, preputium, and glans penis. Immunocytochemistry using NOS antisera corroborated the results of the NADPH-d histochemistry by staining the same two cells in the cerebropleural ganglia. Each of these identifiable nitrergic neurons projects into the ipsilateral pedal ganglion. Because the pedal ganglia play a critical role in the control of locomotion, our results provide morphological evidence suggesting that NO may influence swimming or crawling in Melibe leonina.

Intrinsic choroidal neurons in the duck eye express galanin.

Recently, it has been shown that the choroid of the duck eye harbours approximately 1,000 intrinsic choroidal neurons positive for vasoactive intestinal polypeptide and neuronal nitric oxide synthase. Their connections and functional significance are largely unknown. This study was performed to establish a typical chemical code for these neurons and to define their targets by using immunocytochemistry and confocal laser scanning microscopy. Almost all intrinsic choroidal neurons coexpressed galanin (GAL), vasoactive intestinal polypeptide (VIP), and neuronal nitric oxide synthase (nNOS)/NADPH-diaphorase. A few stained for GAL and/or nNOS only. Among extrinsic ganglia, GAL/VIP/nNOS coexpressing neurons were only found in the pterygopalatine ganglion where they accounted for approximately 30% of the neuronal population. Thus, GAL/VIP/nNOS-positive nerve fibres around branches of the ciliary artery and within the nonvascular smooth muscle stroma of the choroid may originate mainly from intrinsic neurons and to some extent in a subpopulation of pterygopalatine ganglionic neurons exhibiting the same chemical coding. Close contacts of GAL-positive fibres upon intrinsic choroidal neurons may indicate reciprocal connections between them. Thus, intrinsic choroidal neurons may represent peripherally displaced pterygopalatine ganglion neurons forming a local network for regulation of vascular and nonvascular smooth muscle tone in the duck choroid. They may be integrated in the neuronal circuitry controlling intraocular pressure, choroidal thickness, accommodation, and axial bulbus length.

Nitric oxide producing neurons in the monkey and human digestive system.

Nitric oxide has been proposed as an inhibitory transmitter molecule that plays a role in muscle relaxation and vasodilation in the gastrointestinal tract. The present study analyzes the distribution of nitric-oxide-producing neurons in the monkey and human digestive system by means of nicotinamide-adenine-dinucleotide-phosphate-diaphorase histochemistry. This histochemical method is reliable and convenient for the visualization of neuronal nitric-oxide synthase, the enzyme responsible for nitric-oxide generation. In the gastrointestinal tract, nitric-oxide-synthase-related diaphorase activity was present in nerve fibers running throughout the muscular layer (circular > longitudinal) and in numerous ganglion cells and processes in the myenteric plexus of monkeys and humans. Labelled ganglion cells and fibers also were observed in the submucous plexus, although they were much less numerous than those seen in the myenteric plexus. In the submucosa, a few positive fibers were seen around blood vessels. In the mucosa, stained fibers were sparse at the base of the villi and crypts, whereas they were quite abundant in the muscularis mucosae, especially in the small intestine and colon. In the gallbladder (human), labelling was found in ganglion cells and processes of the innermost and outermost ganglionated plexuses. Stained fibers also were distributed to the muscular layer and, less abundantly, to the mucosa and vasculature. Labelled fibers were more abundant in the sphincter of Oddi (human) than in the gallbladder. In the monkey and human pancreas, nicotinamide-adenine-dinucleotide-diaphorase staining was seen mainly in ganglion cells and fibers of intrapancreatic ganglia, and in processes running among acini, around ducts and in the stroma. A moderate density of stained fibers also was distributed to the vasculature, whereas the islets showed few positive processes. Finally, double label experiments performed in the pancreas showed that the vast majority of neurons producing nitric oxide are immunoreactive for vasoactive intestinal peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Laryngeal afferent stimulation enhances Fos immunoreactivity in periaqueductal gray in the cat.

The main functions of the larynx are protection of the airways, respiration, and vocalization. Previous studies have suggested a link between the mechanisms controlling vocalization and afferent feedback from the larynx. We inquired whether stimulation of the laryngeal afferents that run in the internal branch of the superior laryngeal nerve (ISLN) activates neurons of the periaqueductal gray (PAG), a midbrain region implicated in vocalization. We counted the number of neurons expressing Fos, the protein product of the immediate early gene c-fos, in the PAG. The counts were done both in experimental cats after electrical stimulation of the ISLN and nonstimulated controls. We also investigated the possible presence of nitric oxide synthase, an enzyme that synthesizes nitric oxide, in PAG neurons that respond to laryngeal afferent stimulation by double labeling for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase and Fos. Fos expression was significantly greater (P < or = 0.00714) in the lateral and dorsolateral regions of the PAG in the experimental group than in the controls. The Fos-immunoreactive neurons did not contain NADPH-diaphorase, a marker for nitric oxide synthase. Our study suggests that laryngeal afferent stimulation activates neurons in discrete longitudinal columns of the PAG including the regions that have previously been shown to be involved in vocalization, and that these neurons do not contain nitric oxide synthase.

Axonal neurofilament-H immunolabeling in the rabbit retina.

A monoclonal antibody directed at the multiphosphorylated epitope of axonal neurofilament-H (NF-H) was used to label axon-like fibers in the rabbit retina. NF-H-immunopositive fibers were found in the outer plexiform layer (OPL), inner plexiform layer (IPL), and optic fiber layer (OFL). The morphological characteristics of the labeled processes identified those in the OPL as horizontal cell axons and axon terminals and fibers in the OFL as axons of ganglion cells. The NF-H-positive profiles in the OPL formed a subset of horizontal cell processes labeled for calbindin. In the IPL, NF-H-immunoreactive profiles lay at all levels but were detected most often in the middle strata, 2-4. Occasionally, we observed NF-H-immuoreactive processes emerging from the IPL and entering either the GCL or the inner nuclear layer (INL). The labeled fibers in the IPL were typically very thin, less than 1 microm in diameter, and could often be followed for over 1 mm as they ran laterally across the retina. Cell bodies were never labeled by the immunoserum. To identify the NF-H-immunopositive fibers in the IPL, standard immunocytochemical double-labeling techniques were applied, using antibodies directed against several neurotransmitters or modulators thought to be expressed by axon-bearing amacrine cells. The NF-H-positive processes in the IPL were found to correspond to those labeled for tyrosine hydroxylase, somatostatin, substance P, and NADPH diaphorase activity. However, the NF-H labels did not colocalize with those against the vasoactive intestinal peptide-associated protein PHM27. Our results indicate that putative axons in the retina possess the multiphosphorylated NF-H protein found within classic axons in the central nervous system. These results thus support the idea that certain subtypes of amacrine and horizontal cells maintain true axons in the mammalian retina.

Segmental and laminar distributions of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing and neuronal nitric oxide synthase-immunoreactive neurons versus radioassay detection of catalytic nitric oxide synthase activity in the rabbit spinal cord.

The distributions of neuronal nitric oxide synthase-immunoreactive neurons and of nicotinamide adenine dinucleotide phosphate-diaphorase activity were studied in the C6, Th2, L1, L5, S2 and S3 segments and laminae in the rabbit spinal cord and compared with the catalytic nitric oxide synthase activity, determined by monitoring the conversion of [3H]arginine to [3H]citrulline in the same segments and laminae. Morphologically, a heterogeneous population of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing and neuronal nitric oxide synthase-immunoreactive neurons was detected in the superficial and deep dorsal horn and the pericentral region in all segments studied, and in the intermediolateral cell column of the thoracic and lumbosacral segments. A disproportionate distribution of both neuronal categories which had a significantly higher number of nicotinamide adenine dinucleotide phosphate-diaphorase-expressing rather than neuronal nitric oxide synthase-immunoreactive cell bodies was found in all segments. The catalytic nitric oxide synthase activity was distributed unequally in the C6, Th2, L1, L5, S2 and S3 segments, with a comparatively low value in the Th2 segment (70 +/- 5.1 d.p.m./microg protein) in comparison with the S3 segment, where the highest level (140 +/- 5.5 d.p.m./microg protein) was found. A close correlation between the number of neuronal nitric oxide synthase-immunoreactive somata and catalytic nitric oxide synthase activity was revealed in the dorsal horn (laminae I-VI). Whereas a low number of neuronal nitric oxide synthase-immunoreactive somata in laminae VII-X was found in the L5, S2 and S3 segments, the values of catalytic nitric oxide synthase activity in the same laminae and segments were found to be exceedingly high. These findings indicate that the occurrence of many neuronal nitric oxide synthase-immunoreactive fibers (mainly axons), and dense, punctate, non-somatic neuronal nitric oxide synthase immunopositivity in the neuropil staining of the same laminae and segments, can substantially enhance catalytic nitric oxide synthase activity.

Immunohistochemical localization of argininosuccinate synthetase in the rat brain in relation to nitric oxide synthase-containing neurons.

The distribution of the urea cycle enzyme, argininosuccinate synthetase, in the rat brain was determined using immunohistochemistry. This enzyme participates in the only known metabolic pathway for citrulline, its condensation with aspartate to form argininosuccinate, which can then be cleaved to fumarate and arginine. It may thus provide a mechanism to recycle citrulline, formed in the nervous system via nitric oxide synthase activity, back to the nitric oxide precursor, L-arginine. Argininosuccinate synthetase immunoreactivity was detected in discrete populations of neurons throughout the brain. Double-staining with nicotinamide adenine dinucleotide phosphate (reduced form)-diaphorase histochemistry for the localization of nitric oxide synthase demonstrated that argininosuccinate synthetase coexists with nitric oxide synthase in some brain regions. However, many neurons were found that contained one of these two enzymes, but not the other. Thus some nitric oxide synthase-containing neurons appear able to recycle citrulline via argininosuccinate, while others do not. Additional roles for argininosuccinate synthetase in the brain are discussed.

Incipient cauda equina syndrome as a model of somatovisceral pain in dogs: spinal cord structures involved as revealed by the expression of c-fos and NADPH diaphorase activity.

Segmental and laminar distribution of Fos-like immunoreactive, reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-exhibiting and double-labeled (Fos-like immunoreactive and NADPHd-exhibiting) neurons was examined in lower lumbar and sacral segments of the dog spinal cord using the model of multiple cauda equina constrictions. NADPHd histochemistry was used as marker of nitric oxide synthase-containing neurons. The appearance and the time-course of Fos-like immunoreactive, NADPHd and double-labeled neurons was studied at 2 h and 8 h postconstriction characterized as the incipient phase of cauda equina syndrome. The occurrence of Fos-like immunoreactive and NADPHd-exhibiting neurons in fully developed cauda equina syndrome was studied at five days postconstriction. An increase in Fos-like immunoreactivity in superficial laminae (I-II) and an enhanced NADPHd staining of lamina VIII neurons were found. A statistically significant increase in Fos-like immunoreactive neurons was found in laminae I-II and VIII-X 8 h postconstriction, and in contrast, a prominent decrease in Fos-like immunoreactive neurons was found in laminae I-II, accompanied by a statistically significant increase in Fos-like immunoreactive neurons in more ventrally located laminae VII-X at five days postconstriction. Quantitative analysis of laminar distribution of constriction-induced NADPHd-exhibiting neurons revealed a considerable increase in these neurons in laminae VIII-IX 8 h postconstriction and a statistically highly significant increase in NADPHd-exhibiting neurons in laminae VII-X five days postconstriction. Concurrently, the number of NADPHd-exhibiting neurons in laminae I-II was greatly reduced. While a low number of double-labeled neurons was found throughout the gray matter of lower lumbar and sacral segments at 2 h postconstriction, a statistically significant number of double-labeled neurons was found in lamina X 8 h and in laminae VII-X five days postconstriction. The course and distribution of anterograde degeneration resulting five days after multiple cauda equina constrictions are compared with segmental and laminar distribution of Fos-like immunoreactive and NADPHd-exhibiting neurons. Prominent involvement of the spinal cord neurons appearing in the lumbosacral segments at the early beginning and in fully developed cauda equina syndrome results in a Fos-like immunoreactivity and strongly enhanced NADPHd staining of some neuronal pools. Under such circumstances, an early cauda equina decompression surgery is advisable aimed at decreasing or preventing the derangement of the neural circuits in the lumbosacral segments.

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-immunolabeled 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 microM) and NG-nitro-L-arginine (L-NOARG, 10 microM). 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 microM) and had no direct smooth muscle effects per se. 6. Acetylcholine (1 nM-10 microM) 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 mM L-NOARG. 7. In vessel segments contracted with K+ (60 mM), acetylcholine induced concentration-dependent relaxations. When the vessels were incubated with 0.3 mM L-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.

Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite.

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.

Infrequent cellular coexistence of NADPH-diaphorase and calretinin in the neurosecretory nuclei and adjacent areas of the rat hypothalamus.

Colocalization of the calcium-binding protein calretinin and NADPH-diaphorase activity at the cellular level was studied in the magnocellular secretory nuclei of the rat hypothalamus using sequential immunocytochemical and histochemical staining of the same sections. A low degree of colocalization of these markers was observed in certain cellular subpopulations within all the areas considered (supra-optic, paraventricular, circular and both fornicals nuclei and in the hypothalamic area located between the supraoptic and paraventricular nuclei). However, since in the paraventricular nucleus both markers were expressed by different neuronal populations, the coexistence was almost non-existent in some subdivisions of this nucleus. This rare coexistence strongly suggests that NADPH-diaphorase and calretinin are related to different functions shared by restricted hypothalamic neuronal populations.

Cytochrome b558 (p22phox) in the guinea-pig adrenal medulla.

Paraganglionic cells are sensitive to hypoxia, and the involvement of a plasmalemmal cytochrome b558-like protein in oxygen sensing by these cells has been suggested, but neither the identity of the immunoreactive protein detected by immunohistochemistry nor its anticipated subcellular (i.e., plasmalemmal) localization were directly proven. Thus, we extended these studies to the largest paraganglion, i.e., the adrenal medulla, in the guinea-pig, which, due to its size and accessibility, allowed us to address both of these issues utilizing antisera raised against synthetic peptides of the small (22 kD) subunit of cytochrome b558, p22phox. Cytochrome b558 was originally identified in granulocytes and macrophages, and antisera against this phagocyte p22phox were utilized. Immunoreactivity to p22phox was observed in all adrenal medullary endocrine cells, and the identity of the immunoreactive protein to the small cytochrome b558-subunit was confirmed by Western blotting. Immuno-electron microscopy of ultrathin cryosections and of resin-embedded tissue demonstrated its subcellular localization in the dense core vesicles of endocrine A-cells but not in the plasma membrane. In conclusion, the present study documents the presence of the small subunit of cytochrome b558 in guinea-pig adrenal medullary cells, but its subcellular vesicular localization does not support the initial interpretation of cytochrome b558 serving as a plasmalemmal oxygen sensor.

Nitroxergic autonomic neurones in rat spinal cord.

We have used a polyclonal monospecific antibody to rat cerebellum nitric oxide synthase type I (NOS-I, 160 kD) in combination with reduced NADPH-diaphorase histochemical reaction (NADPH-d) to verify colocalization of both NOS protein and NOS enzymatic activity in the rat spinal cord autonomic system. Strong immunoreactivity (IR) of NOS-I was clearly detected in the four main thoracolumbar autonomic nuclei in spinal cord layers of Rexed's laminae VI, VII and X. In all labelled neurones, NOS-I-IR colocalized with NADPH-d activity, suggesting coexistence of brain-specific NOS-I-like protein and its associated enzyme activity. For these neurones the new term 'nitroxergic' (i.e. NO-generating) neurones appears to be justified. Spinal cord nitroxergic neurones are part of a NO-mediated signal transduction pathway for control of the sympathetic 'outflow' to various peripheral target organs.