Nitric oxide (NO) modulates diverse functions of polymorphonuclear neutrophils (PMNs), but localization of NO synthase (NOS) and identification of its interacting proteins remain the least defined. The present study discerns subcellular distribution of NOS and caveolin-1, a prominent NOS-interacting protein in rat PMNs. Localization of NOS was explored by confocal and immunogold electron microscopy, and its activity was assessed by L-[3H] arginine and 4,5-diaminofluorescein diacetate (DAF-2DA). Reverse transcriptase-polymerase chain reaction using NOS primers and Western blotting demonstrated the presence of neuronal NOS (nNOS) and inducible NOS (iNOS) in PMNs. Immunocytochemical studies exhibited distribution of nNOS and iNOS in cytoplasm and nucleus, and L-[3H] citrulline formation and DAF fluorescence confirmed NOS activity in both fractions. NOS activity correlated positively with calmodulin concentration in both of the fractions. nNOS and iNOS colocalized with caveolin-1, as evidenced by immunocytochemical and immunoprecipitation studies. The results thus provide first evidence of nNOS and iNOS in the nuclear compartment and suggest NOS interaction with caveolin-1 in rat PMNs.

Recent studies from our laboratory have demonstrated that ascorbate potentiated enzymatic synthesis of nitric oxide (NO) from polymorphonuclear leukocytes (PMNs). NO is known to modulate various function of PMNs such as chemotaxis, adherence, aggregation, and generation of reactive oxygen species (ROS). The role of ascorbate in the PMN phagocytosis, ROS generation, and apoptosis was thus evaluated in the present study. Ascorbate and its oxidized and cell-permeable analog, dehydroascorbate (DHA), did not affect the phagocytosis but enhanced ROS generation and apoptosis following treatment with Escherichia coli or arachidonic acid. A detailed investigation on the DHA-mediated response indicated that inhibitors of DHA uptake, reduced nicotinamide adenine dinucleotide phosphate oxidase, NO synthase, or ROS scavengers attenuated ROS generation. In DHA-treated cells, enhanced generation of peroxynitrite was also observed; thus, ascorbate-mediated ROS and reactive nitrogen species generation might mediate cytotoxicity toward the ingested microbes and subsequently, augmented PMN apoptosis. Results of the present study have helped in delineating the role of ascorbate in the modulation of NO-mediated ROS generation from PMNs.

Neutrophils play a key role in innate immune responses against foreign intrusion and influence the subsequent instigation of adaptive immune response. Nitric oxide (NO) synthesized by neutrophil nitric oxide synthase (NOS) profoundly modulates their diverse physiological responsibilities furthermore encompassing pathological implications. Neutrophils are the active participants in diverse inflammatory and cardiovascular disorders but neutrophil nitric oxide synthase (NOS) remains enigmatic on various aspects. This review focuses on inducible NOS (iNOS) and makes an attempt to address its potential impact in neutrophil pathophysiology, their differentiation, functionality, and survival. We described the scenario from its expressional modulation, by pro-and anti-inflammatory cytokines governing the extent and duration of neutrophil immune response, to iNOS catalysis, the intracellular compartmentalization, and protein-protein interactions determining its microenvironment, activity and its contribution as a potential signaling protein apart from its role as signal transducer. Further, the relevance of investigating the unexplored facets of iNOS biology in neutrophils and possible prototypes of iNOS regulation is also exemplified in related cellular systems. K E Y W O R D Sinducible nitric oxide synthase, neutrophils, protein interaction, regulation, subcellular localization INTRODUCTIONSince its discovery as endothelium-derived growth factor, NO has been related to a broad spectrum of signaling pathways in diverse pathophysiological, immunomodulatory, 1-3 and pharmacological responses.NO regulates functional attributes of neutrophils modulating their chemotactic, phagocytic, migratory, and apoptotic activities 4 as elaborated in Fig. 1. Neutrophils generate NO at a rate of (10-100) nmoles /5 min/10 6 cells. 5 These comprise 60-70% of the total peripheral leukocytes in humans, contribute significantly to the circulating levels, encompassing a widespread impact on cardiovascular homeostasis.Being capable against invading pathogens, a tumor cell, and alloantigens, NO is an asset to these immunocompetent cells, adding more to their cytotoxic battalion. It has also been demonstrated that NO mediates neutrophil extracellular traps (NET) release at the site of Abbreviations: AL, arginosuccinate lyase; AS, arginosuccinate synthase; Asc, ascorbate; BH 4 , tetrahydrobioptein; CaM, calmodulim; CGD, chronic granulomatous disease; DHA, dehydroascorbate; ETC, electron transport chain; Hsp, heat shock protein; I B, inhibitor kappa B; NLS, nuclear localization signal; PDZ, PSD-95/Disc large/ZO-1; PKC, protein kinase C; RNI, reactive nitrogen intermediates; ROCK, Rho-associated coiled coil containing protein kinase; TGF, tumor growth factor inflammation / infection by augmenting free radical generation. 6 Study by Napimoga et al. 7 depicted that peroxisome proliferator-activated receptor-(PPAR-) reduces neutrophil migration to the acute inflammatory mesenteric lesion as a result of reduction of rolling and adhesion activities of neu...

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Tenebrio molitor (Tenebrionidae) In Tenebrio six apposed thin Malpighian tubules arise from a common stem (fig. I), covered with the peritoneum, which later forms the perinephric membrane of the system and encloses the rectum; after entering the membrane the tubules again become separate and run inside the perinephric chamber in a serpentine fashion; at regular intervals they are attached to the perinephric membrane. The complex formed by the rectum and the reassociated Malpighian tubules in intimate relationship appears to constitute a system; it is therefore necessary to describe the histology of the gut and the Malpighian tubules.In the neighbourhood of the common stem the cytoplasm of the six apposed tubules is not thick and the brush border is very feeble. The rectal sac and the reassociated tubules can be regarded as consisting of three zones. In the anterior, the rectal epithelium shows six clear rectal pads, the cells of which have big round nuclei, and is surrounded with thick circular and longitudinal muscles. The tubules surrounding this region are thin and hardly convoluted, except at the posterior end. The brush border of the tubules is small, and mitochondria are not seen ( fig. 1). In the middle zone ( fig. 2) the tubules are very convoluted, come close to the rectal epithelium (which has bigger cells) and show leptophragmata at the attachment with the perinephric membrane. There is a musculature between the tubules and the gut epithelium, which is full of mitochondria and has a folded basement membrane. These large epithelial cells, with numerous mitochondria, probably concerned in the active transport of water and inorganic ions, are referred to throughout this paper as "active epithelium". The hind zone ( fig. 2) is the posterior part of the rectal sac, lying just before the hinge to which the perinephric membrane is attached by connective tissue. In this hind part the rectal epithelium is highly modified; it is full of mitochondria and the nuclei lie towards the basement membrane, which is very much folded. The reassociated tubules come very close to the epithelium, and here the musculature is greatly reduced. The tubules surrounding this zone are very convoluted and well developed. Their brush border contains closely packed vesicles, which are full of rod-like mitochondria. The cytoplasm is quite thick here, and the tubules are surrounded by a clearly defined basement membrane. Posterior to the active zone of epithelium there is a hinge (fig. 2, hng) (to which the perinephric tissue is attached), which acts as a valve between the rectal sac and the rectal canal; it is formed of yellow hard cuticle, in which the epithelial cells of the gut are missing. Just behind this hinge is the rectal canal, which is covered with a thick musculature, especially conspicuous under the hinge, the function of which is probably to retain the excreta in the rectal sac for removal of water.A leptophragma occurs at the centre of each prominent swelling found in the cryptonephridial sheath, and at each of these po...