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      Aging-Related Increased Expression of Inducible Nitric Oxide Synthase and Cytotoxicity Markers in Rat Hypothalamic Regions Associated with Male Reproductive Function

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          Abstract

          We have previously demonstrated that the inducible nitric oxide synthase (iNOS) protein and total NOS activity increase in the hypothalamus and other regions of the male rat brain during aging. We have now tested the hypothesis that increased iNOS results in excessive nitric oxide (NO) and peroxynitrite production, and leads to increased apoptosis in CNS cells, including the GnRH and oxytocin hypothalamic neurons involved in the control of male reproductive function. Young (3-month-old) and old (24-month-old) male Brown Norway rats (n = 6) were perfused with 4% formalin. Adjacent coronal paraffin-embedded sections (5 µm) of preoptic area (POA), supraoptic nucleus (SON), paraventricular nucleus (PVN), and arcuate nucleus (ARC) of the hypothalamus were immunostained with antibodies for iNOS, neuronal NOS (nNOS), and nitrotyrosine (a marker of peroxynitrite formation). The intensity of immunostaining was measured using a densitometric image analysis system. Apoptosis was determined by the TUNEL assay. Double immunofluorescence staining with confocal laser scanning microscopy was used for co-localization studies. A significant increase in the iNOS immunostaining measured as optical density (OD) was found in the old compared to the young animals (SON: 0.32 ± 0.02 vs. 0.23 ± 0.03, p < 0.05; PVN: 0.34 ± 0.03 vs. 0.07 ± 0.05, p < 0.001; POA: 0.18 ± 0.02 vs. 0.01 ± 0.02, p < 0.001). Aging did not affect nNOS expression. Nitrotyrosine was elevated in the hypothalamic regions of old compared to young rats (SON: 0.32 ± 0.05 vs. 0.10 ± 0.04, p < 0.05; PVN: 0.32 ± 0.04 vs. 0.13 ± 0.03, p < 0.01; POA: 0.72 ± 0.06 vs. 0.03 ± 0.003, p < 0.001). Increased nitrotyrosine was accompanied by an elevation of the apoptotic index in the old rats (SON: 11.01 ± 3.33 vs. 0.57 ± 0.50, p < 0.001; PVN: 3.08 ± 1.12 vs. 0.42 ± 0.32; POA: 6.60 ± 1.93 vs. 0.18 ± 0.17, p < 0.01; ARC: 0.001 ± 0.0001 vs. 4.33 ± 2.33). iNOS staining co-localized with GnRH and oxytocin staining. In conclusion: The aging-related iNOS increased expression in the hypothalamus of the male rat affects regions known to control the synthesis and release of GnRH (POA, ARC) and oxytocin (PVN, SON), and the factors regulating penile erection (POA, and PVN). These observations suggest that iNOS may play a role in the reduction in GnRH and oxytocin neuronal secretion resulting in reproductive dysfunctions such as lowered serum testosterone, hypospermatogenesis, and diminished copulatory function in the aging male animal.

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          Most cited references 5

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          Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes.

          Nitric oxide (NO) is a potent inhibitor of apoptosis in many cell types, including hepatocytes. We and others have described NO-dependent decreases in caspase activity in cells undergoing apoptosis. However, previous work has not determined whether NO disrupts the proteolytic processing and thus the activation of pro-caspases. Here we report that NO suppresses proteolytic processing and activation of multiple pro-caspases in intact cells, including caspase-3 and caspase-8. We found that both exogenous NO as well as endogenously produced NO via adenoviral inducible NO synthase gene transfer protected hepatocytes from tumor necrosid factor (TNF) alpha plus actinomycin D (TNFalpha/ActD)-induced apoptosis. Affinity labeling with biotin-VAD-fmk of all active caspase species in TNFalpha-mediated apoptosis identified four newly labeled spots (activated caspases) present exclusively in TNFalpha/ActD-treated cells. Both NO and the caspase inhibitor, Ac-DEVD-CHO, prevented the appearance of the four newly labeled spots or active caspases. Immunoanalysis of affinity labeled caspases demonstrated that caspase-3 was the major effector caspase. Western blot analysis also identified the activation of caspase-8 in the TNFalpha/ActD-treated cells, and the activation was suppressed by NO. Furthermore, NO inhibited several other events associated with caspase activation in cells, including release of cytochrome c from mitochondria, decrease in mitochondrial transmembrane potential, and cleavage of poly(ADP-ribose) polymerase in TNFalpha/ActD-treated cells. These findings indicate the involvement of multiple caspases in TNFalpha-mediated apoptosis in hepatocytes and establish the capacity of NO to inhibit not only active caspases but also caspase activation.
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            Oxytocin stimulates the release of luteinizing hormone-releasing hormone from medial basal hypothalamic explants by releasing nitric oxide.

            Oxytocin induces mating behavior in rats of both sexes. Previous experiments revealed that progesterone-induced sex behavior in ovariectomized, estrogen-primed rats was caused by release of NO from NOergic neurons that stimulated the release of luteinizing hormone-releasing hormone (LHRH). The LHRH activated brain-stem neurons that initiated the lordosis reflex. We hypothesized that oxytocin might similarly release NO in the medial basal hypothalamic region that would stimulate release of LHRH into the hypophyseal portal vessels to release luteinizing hormone. To investigate this hypothesis, medial basal hypothalamic explants were preincubated in Krebs-Ringer bicarbonate buffer for 30 min, followed by a 30-min incubation in fresh Krebs-Ringer bicarbonate buffer containing the compounds to be tested. Oxytocin stimulated LHRH release 3- to 4-fold at the lowest concentration tested (10(-10) M). Values remained at a plateau as the concentration was increased to 10(-7) M and then declined in a concentration-dependent manner, so that there was no stimulation with a concentration of 10(-5) M. Oxytocin (10(-7) M) stimulated release of prostaglandin E2 into the medium, a finding consistent with a role of NO in the response. That NO indeed mediated the action of oxytocin was supported by blockade of the action of oxytocin by the competitive inhibitor of NO synthase (NOS), N(G)-monomethyl-L-arginine (300 microM). Furthermore, oxytocin (10(-9) to 10(-7) M) activated NOS as measured at the end of the experiments. Oxytocin appeared to act to stimulate norepinephrine terminals in the medial basal hypothalamus, which activated NOS by alpha1-adrenergic receptors, because prazocine, an alpha1 receptor blocker, inhibited the LHRH-releasing action of oxytocin. Finally, incubation of neural lobe explants with sodium nitroprusside, a NO releasor, revealed that nitroprusside (300-600 microM, but not 900 microM) inhibited oxytocin release. Therefore, the NO released by oxytocin also diffuses into the oxytocin neuronal endings and inhibits oxytocin release, forming a negative feedback loop. The results indicate that oxytocin is important not only in induction of mating, but also in stimulating LHRH release with subsequent luteinizing hormone discharge that plays a crucial role in reproduction.
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              Elevated oxidative stress in models of normal brain aging and Alzheimer's disease

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                Author and article information

                Journal
                NEN
                Neuroendocrinology
                10.1159/issn.0028-3835
                Neuroendocrinology
                S. Karger AG
                0028-3835
                1423-0194
                2001
                July 2001
                06 July 2001
                : 74
                : 1
                : 1-11
                Affiliations
                aDepartment of Urology, UCLA School of Medicine, Los Angeles, Calif.,Divisions of bUrology and cEndocrinology, Harbor-UCLA Medical Center and Research and Education Institute, Torrance, Calif., USA
                Article
                54665 Neuroendocrinology 2001;74:1–11
                10.1159/000054665
                11435753
                © 2001 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 5, References: 55, Pages: 11
                Categories
                Reproductive Neuroendocrinology

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