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Endocrinology Vol. 139, No. 7 3254-3261
Copyright © 1998 by The Endocrine Society

ARTICLES

Spontaneous Expression of Inducible Nitric Oxide Synthase in the Hypothalamus and Other Brain Regions of Aging Rats1

Dolores Vernet, Juan Jose Bonavera, Ronald S. Swerdloff, Nestor F. Gonzalez-Cadavid and Christina Wang

Departments of Urology and Medicine, UCLA School of Medicine, Harbor-UCLA Medical Center, Torrance, California 90509

Address all correspondence and requests for reprints to: Nestor Gonzalez-Cadavid, Ph.D., Harbor-UCLA Medical Center, Division of Urology, Department of Surgery, Bldg. F-6, 1000 West Carson St., Torrance, California 90509.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our laboratory has demonstrated that aging in Brown-Norway rats is associated with decreased LH pulse amplitude and reduced GnRH and LH responsiveness to excitatory amino acids (EAA), presumably through the NMDA receptor (NMDAR). Nitric oxide (NO) is a neurotransmitter postulated to be involved in hypothalamic synaptic events required for normal GnRH regulation through the activation of neuronal nitric oxide synthase (nNOS). Paradoxically, excessive stimulation of nNOS by NMDAR or the expression of inducible nitric oxide synthase (iNOS) can lead to supraphysiological levels of NO acting as effector of apoptosis with resultant decreased regional neuronal function. The aims of this study were to determine: 1) whether aging in the preoptic area/medial basal hypothalamus is associated with altered NO synthesis; 2) the possible roles of the NMDAR/nNOS cascade and iNOS in this process; and 3) whether alterations in the levels of NOS isoforms are specific to this region of the brain. Brown Norway male rats (N = 5) at ages 1 (immature), 3 (adult), and 24 (old) months, were used for measuring NMDARs in hypothalamic membranes by the binding of a (3H)-NMDAR ligand. Another series of the same age groups of rats (N = 9) were used to determine by Western blot the contents of NMDAR, nNOS, and iNOS in the hypothalamus, and only iNOS in the frontal and parietal cortex, and cerebellum. NOS activity was measured in the hypothalamus by the arginine/citrulline assay. A significant decrease of NMDA analog binding was found in the hypothalamus from old rats as compared with adult (-66%) and immature animals (-57%), accompanied by a reduction in NMDAR content (-34% and -46%, respectively). NOS activity in the hypothalamus was 67% and 100% higher in old rats as compared with the other two groups, although no significant differences were observed in nNOS content. However, hypothalamic iNOS increased 3.8- and 7.6-fold in old rats, as compared with adult and immature, respectively. This increase in hypothalamic iNOS was paralleled by a rise of iNOS in other brain regions of old rats as compared respectively to adult and immature animals: 3.9- and 12.8-fold, in the frontal cortex; 2.8- and 2.5-fold, in the parietal cortex; and 3.1- and 4.8-fold, in the cerebellum. These results show that aging in this rat model is associated with high NO synthesis in the hypothalamus and other regions of the brain, which is independent of the NMDAR/nNOS cascade. We speculate that increased brain levels of iNOS may lead to neurotoxicity, which may be involved in GnRH impaired pulsatile secretion, as well as acting as a possible inducer of age associated neuronal loss in cognitive related brain areas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MEN, reproductive aging is characterized by a primary testicular dysfunction coupled with hypothalamic-pituitary dysregulation characterized by abnormal pulsatile secretion of GnRH (1, 2, 3), and a reduction of the amplitude but not of frequency of LH pulses. The aging-related changes in pulsatile GnRH secretion in men are also found in old Brown Norway rats (4, 5), a model used for studies of reproductive aging because it manifests both primary and secondary testicular failure (6, 7). Our group has shown that these animals have, similar to men, a decreased LH pulse amplitude and reduced GnRH and gonadotropin responsiveness to excitatory amino acid (EAA) agonists both in vivo and in vitro (5, 8). The decreased GnRH-LH responsiveness to EAA may be a component of the complex interplay by several neuropeptides and neurotransmitters in the regulation of GnRH content and pulsatile secretion (9, 10). In Brown Norway and Fischer 344 rats (4, 11, 12, 13), and in mice (14), GnRH mRNA and peptide content were demonstrated to be significantly reduced in the medial preoptic area of the hypothalamus in old compared with young animals. In addition to the decrease in hypothalamic GnRH neurons, there was a decline in neuronal activity, as evidenced by down-regulation of the expression of immediate early gene products in GnRH neurons in older female rats when compared with younger animals (15).

The binding of excitatory amino acids (EAA) such as glutamate or aspartate to the N-methyl-D-aspartate receptor (NMDAR) results in both the stimulation of GnRH release and the modulation of its pulsatile pattern in the hypothalamus (16, 17, 18, 19, 20, 21). The NMDARs have been implicated in the central activation of the reproductive axis, both at puberty and at the reinitiation of gonadal activity in seasonal breeders (16, 21). These ionotropic glutamate receptors are considered to be the main neurotransmitter receptors mediating fast synaptic excitation in the CNS (22, 23), through the opening of Ca2+ channels triggering a series of metabolic cascades. One of these pathways is the stimulation of neuronal nitric oxide synthase (nNOS) that enhances the synthesis of nitric oxide (NO) (23), which is assumed to be the mediator of at least some of the neuroendocrine effects of glutamate in the CNS, and specifically of GnRH and LH secretion (9, 10, 16, 21, 24, 25, 26, 27, 28, 29). In the prepubertal and adult hypothalamus, nNOS is the predominant NOS isoform (30) and colocalizes with or is adjacent to NMDAR and GnRH neurons in the medial preoptic area (28, 31).

It is conceivable that in addition to controls exerted by neuropeptides and catecholamines, the hypothalamic NMDAR/NO cascade may play a role in the aging-related alteration of the GnRH pulsatile secretion by a decrease in NO synthesis due to a reduction in EAA/NMDAR binding that would diminish the release of GnRH (8, 16, 20, 22, 24, 32, 33). NMDARs have been found to be decreased in the forebrain of old animals (34, 35, 36, 37, 38, 39), and aging is accompanied by a reduction of constitutive NOS activity in other organs (40, 41). No information is available on the effect of aging on the levels of NMDAR and NOS in the hypothalamus, although we have shown that the concentration of glutamate in this organ is reduced in old rats (8).

An alternative hypothesis for NOS involvement in age-associated hypothalamic dysfunction could be that aging is accompanied by an excessive synthesis of NO and the accumulation of cytotoxic levels of its metabolites (42, 43, 44, 45, 46), leading to an increase in neuronal apoptosis. This process may affect hypothalamic neurons, including those that secrete GnRH, among other neurons involved in functions impaired by aging. This may occur as a result of an excessive stimulation of nNOS by a high rate of activation of NMDARs (16, 22, 23). However, another mechanism for higher NO levels in the old brain tissue may be the spontaneous expression of the inducible NOS (iNOS), the NOS isoform (16, 47) induced during the immune defense, or in autoimmune, inflammatory, or degenerative processes. High NO levels may also result in feedback inhibition of NOS activity and/or expression (9), a process that has been shown to occur in other systems (23, 48), and also elicit a direct inhibition of GnRH synthesis. The latter may occur through the interaction with an element in the GnRH gene promoter that is repressed by the NO-mediated cGMP signal transduction pathway (49).

Although, under physiological conditions, iNOS is virtually undetectable in organs of adult laboratory animals, and specifically in the hypothalamus and other regions of the brain (9, 16, 17, 21), it may be expressed at moderate or high levels in these tissues (50, 51, 52, 53, 54, 55) after exogenous cytokine stimulation and in infectious or inflammatory processes. The observations that aging in rats and humans is associated with an increase in iNOS inducers such as circulating and tissue cytokines (44, 56, 57), and of the apoptotic index (58, 45, 46), suggest that a spontaneous expression of iNOS may occur in the aging hypothalamus and lead to higher NOS activity.

These alternative hypothesis for NOS or NO involvement in altered hypothalamic function have been investigated in the present work by determining NMDAR/nNOS and iNOS levels and activity, and verifying whether iNOS is expressed in other regions of the rat brain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
NADPH, protease inhibitors, N-({omega}-nitro-L-arginine methyl esther (L-NAME), and other reagents were from Sigma Chemical Co., St. Louis, MO. L-[2,3,4,5-3H]arginine mono hydrochloride (specific activity: 35–70 Ci/mmol) was purchased from Amersham Corp. (Arlington Heights, IL) and was purified in our laboratory by column chromatography. CGP 39653, [propyl-2,3-3H] (specific activity: 34 Ci/mmol) was purchased from DuPont NEN (Boston, MA). The antihuman nNOS and iNOS, and the antirat NMDAR-2B monoclonal antibodies were from Transduction Laboratories (Lexington, KY). The polyacrylamide ready gels, prestained SDS-PAGE standards, miniprotean II electrophoresis cell, and mini trans-blot electrophoretic transfer cell were from Bio-Rad Laboratories (Hercules, CA). The horseradish peroxidase-linked secondary antibodies (antimouse IgG), the Hybond ECL Western membrane, and the ECL Western blotting kit were from Amersham, Life Science Division. The BCA protein assay kit was from Pierce (Rockford, IL). The densitometric Scan Analysis program for immunoblots was from BioSoft (Cambridge, UK), and was applied to gel images generated with the OneScanner/Ofoto scanning system from Apple Inc. (Cupertino, CA).

Animals
Immature (1 month old), adult (3 month old), and old (24 months old) male Brown Norway rats (7, 8) were obtained through the NIH National Institute of Aging and supplied by Charles River Laboratories (Wilmington, MA). Animals were maintained under controlled temperature and lighting (lights on from 0500 h to 1900 h) and were treated according to the recommendations of the American Veterinary Medical Association, and the experimental protocols were approved by the Harbor-UCLA REI Animal Care and Use Review Committee. Tissue was dissected either after decapitation (NMDAR determinations) or during thiopental anesthesia (NOS determinations), between 0900 and 1000 h. The preoptic area/medial basal area of the hypothalamus, frontal cortex, parietal cortex, and cerebellum, were obtained and stored at -80 C.

Measurement of [3H]CGP 39653 binding to the NMDAR
A standard procedure was applied (59, 60). Briefly, the hypothalamus was homogenized in 50 vol of 5 mM Tris-HCl buffer, pH 8 (at 4 C), and membranes were sedimented at 48,000 x g for 10 min. Pellets were resuspended and incubated in buffer containing 10 mM EDTA at 37 C (10 min), rinsed twice by centrifugation, stored at -]70 C, thawed, centrifuged, washed twice more, and finally resuspended in 12.5 vol of buffer. Binding was measured in triplicate in 0.1 ml homogenate in 1 ml containing 2 nM [3 H]CGP 39653, with or without 1 mM L-glutamate, for 60 min at 0 C. Bound radioactivity was determined by filtration through glass fiber discs, correcting by unspecific binding.

Measurement of NOS activity
NOS activity in brain homogenates was determined as previously described (40, 61). Briefly, each tissue was weighed and homogenates were prepared in either 10 vol (cerebellum) or 6 vol (all other regions) of cold medium containing 0.32 M sucrose/20 mM HEPES, pH 7.2/0.5 mM EDTA/1 mM dithiothreitol, and protease inhibitors (3 µM leupeptin, 1 µM pepstatin A, 1 mM phenylmethyl sulfonylfluoride), using the Polytron homogenizer (Brinkmann, Lucerne, Switzerland). The postmitochondrial (cytosol) and particulate fractions were separated by centrifugation at 12,500 x g for 60 min at 4 C. The cytosol fraction was passed through Dowex AG50WX-8 (Na+) resin to remove endogenous arginine, and 50-liter aliquots were incubated in triplicate for 45 min at 37 C as indicated, in the presence of 2 µCi/ml resin-purified (3H) L-arginine, 2 mM NADPH, 0.45 mM Ca2+, and 100 (M L-arginine. After eliminating the residual (3H) L-arginine through the resin, (3H) citrulline was counted in the trichloroacetic acid ether-extracted supernatant. Determinations were in triplicate. All values were corrected by the radioactivity eluted in time zero incubations, and expressed per gram of original tissue.

Measurement of NOS and NMDAR content by Western blots
Equal amounts of protein (30 µg) of the postmitochondrial fraction from the rat brain regions were run on 7.5% polyacrylamide SDS gels, and the proteins were transferred to a nitrocellulose membrane for 16 h at 30 V, followed by 0.5 h at 100 V (61, 62). The transfer efficiency was controlled by gel staining with Coomassie blue. Prestained protein markers (48–199 kDa) were always run in each gel. The immuno-detection on the Western blot was carried out with an affinity chromatography purified primary antibody consisting on the mouse monoclonal antibody against a 22.3-kDa fragment of the carboxy terminus of human cerebellum nNOS (1 h, 1:1,000 dilution). The secondary antibody was an antimouse IgG (rabbit) linked to horse radish peroxidase and the incubation (1:2,000 dilution) was carried out for 1 h. The reactive bands were detected with a luminol-based kit. The cytosol from rat cerebellum was always used as a positive control.

In the case of iNOS, the primary antibody consisted of a mouse monoclonal against a 20-kDa protein fragment containing amino acids 961-1144 (carboxy-terminus) of mouse iNOS (1:1,000). The secondary antibody and the detection procedure on the postmitochondrial fraction were as described above for the monoclonal nNOS. Samples were run on PAGE as above (80 µg protein for hypothalamus, 30 µg for frontal cortex, 60 µg for cerebellum and cortex) and submitted to Western blot analysis. The cytosol from rat penile smooth muscle cells (RPSMC) which had been induced with bacterial lypolysaccharide (LPS) and interferon-{gamma} (47, 63) was used as control.

In the case of NMDAR, the primary antibody consisted on a mouse monoclonal against an 18.2-kDa protein fragment containing amino acids 892-1051 (middle) of rat NMDAR subunit 2B (1:500) (64). Samples from the membrane extracts (see above) were run (30 µg/lane) and determinations were as above.

The quantitative determination of band intensities (61, 62) was carried out by submitting each luminol-reacted membrane to several x-ray exposure times and selecting the one(s) falling within the film response range. The films were then scanned and each band density evaluated by densitometry with an adequate program.

Statistical analysis
Experimental values were expressed as mean ± SEM for the number of separate animals indicated in each case. The normality distributions of the data were established using the Wilk-Shapiro normality test. Multiple comparison among the three age groups were analyzed by a single-factor ANOVA. Fisher’s protective least significant differences (pLSD) test was performed to determine whether differences between groups were significant, at levels of significance of 95% (P < 0.05) and 99% (P < 0.01)


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Down-regulation of NMDA receptors during aging
To determine whether the number of NMDA receptors is altered in the hypothalamus of old rats, leading to a possible impairment of NOS-activated pathways, a ligand binding assay was applied with an NMDA agonist. The binding of 3H-CGP to a suspension of crude synaptosomal vesicles was measured in tissue from each individual animal within the three age groups. Data were obtained in triplicate and corrected by nonspecific binding in the presence of excess unlabeled glutamate. Because of the relatively small amount of hypothalamic tissue available per rat and the total number of assays per animal, a saturation curve and Scatchard plot could not be obtained for each sample. However, data from the literature (59, 60) and preliminary experiments showed that the single selected 3H -CGP concentration was saturating for the amount of membrane protein assayed.

Figure 1Go, top, shows that the membrane-associated binding activity for the NMDA agonist in the hypothalamus from old rats was 66 and 57% lower when compared with the adult and immature animals, respectively. This decrease is not due to differences in the endogenous excitatory amino acid pool because the membrane preparation procedure assures the elimination of all traces of vesicle contents by cycles of freezing/thawing/washing.



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  		Figure 1. Effects of aging on the levels of NMDA receptors in the rat hypothalamus. Top panel, Specific binding activity of a crude 48,000 g/10 min hypothalamic membrane fraction for NMDAR ligands was estimated in triplicate with 2 nM [3H] CGP, after disruption and exhaustive rinsing (n = 4). Middle panel, autoradiography of the 180-kDa band on a typical Western blot of the membrane fractions analyzed above (30 µg protein/lane) with an antibody against NMDAR subunit 2B and visualization with a luminol reaction. Bottom panel, Mean intensity of the respective bands determined by densitometry (n = 3). Values represent means ± SEM. Different letters in superscripts denote significant differences between those groups (P < 0.05).

 
The results obtained by measuring binding activity were confirmed by the determination of NMDA receptor content with a Western blot assay similar to the one applied for nNOS, except that the immunodetection was performed with an antibody against subunit 2B of the human brain NMDA receptor. Visual inspection of a film corresponding to a typical Western blot (Fig. 1Go, middle) indicates that the intensity of the 180-kDa band is reduced in the tissue membranes obtained from the hypothalamus of old animals. The densitometric analysis of all specimens analyzed on different gels (Fig. 1Go, bottom) showed that the reduction of NMDA receptor in the old rats as compared with adult and immature animals was 34 and 45%, respectively, agreeing with the binding activity, although the reductions in NMDAR content are slightly lower than the binding estimations.

Increase of NOS activity during aging without affecting nNOS content
NOS activity was measured by the L-arginine/citrulline conversion assay in the 12,500 g/60 min supernatant obtained from the hypothalamus of 1-, 3-, and 24-month-old rats, designated as pubertal, adult, and old animals, respectively. This postmitochondrial fraction in many tissues (including the CNS) contains all the iNOS, most of nNOS, and a variable fraction of eNOS (23, 61, 62). Figure 2Go, top, shows that there are significant increases of soluble NOS activity in the old rats as compared with the adult and immature animals, of 67% and 100%, respectively.



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  		Figure 2. Effect of aging on NOS activity and nNOS content in the rat hypothalamus. Top panel, NOS activity was measured in the 12,500 g/60 min supernatant by the arginine-citrulline conversion assay (n = 9). Middle panel, autoradiography of the 155- and 135-kDa bands on a typical Western blot of the postmitochondrial supernatants analyzed above and additional ones (30 µg protein/lane) with an antibody against human nNOS and visualization with a luminol reaction. Bottom panel, Mean intensity of the respective top bands representing the full length nNOS determined by densitometry (n = 8). Values represent means ± SEM. Different letters in superscripts denote significant differences between those groups (P < 0.01); NS, P: 0.06.

 
Because nNOS was considered to represent most of NOS activity in the hypothalamus (16, 21, 30), aliquots containing equal amounts of protein from the soluble fractions from each individual animal were submitted to Western blot analysis to determine nNOS content. A monoclonal antibody against human nNOS and a luminol-based reaction were applied for detection. This antibody cross-reacts with rat nNOS, has been abundantly characterized for its specificity (61, 62), and detects with equal efficiency nNOS variants such as the recently cloned PnNOS (65).

Figure 2Go, middle panel, shows the film corresponding to a typical x-ray with tissue extracts from three rats out of the nine analyzed per age group, and rat cerebellum as a positive control. The 155-kDa band contains the full-length nNOS for either nNOS or PnNOS and the 135-kDa fainter band is assumed to be a product of alternative splicing or promoter usage (65). The intensity of the top bands appeared similar when comparing age groups, and this was corroborated by densitometric analysis standardized against the signal of the positive control in each immunoblotted membrane (Fig. 2Go, bottom panel). No significant difference was found in the nNOS content values among the three age groups.

Up-regulation of iNOS expression during aging
The elevation of NOS activity in the hypothalamus of old rats without a parallel increase in the content of nNOS suggests that either NOS activity is stimulated per se, or that the expression of other NOS isoforms is increased. To investigate the latter possibility, the effect of aging on the content of iNOS in the hypothalamus was determined. Figure 3Go (top) shows the film corresponding to a typical Western blot, where each gel corresponds to the fractionation and immunodetection of 2–3 specimens from each age group. The iNOS 130-kDa band is virtually nondetectable in the tissue from immature animals and only faintly visible in the tissue from adult rats. In contrast, the iNOS band is clearly present in the hypothalamus from old rats, indicating a significant level of expression. The top band arises from cross-reactivity of the monoclonal anti iNOS antibody with nNOS, as indicated by separate experiments with rat cerebellum extracts and by its absence in the induced RPSMC positive control, which lacks nNOS. The intensity of this top band does not increase with age in the hypothalamus, or in the other brain regions (see Fig. 4Go).



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  		Figure 3. Effect of aging on iNOS levels in the rat hypothalamus. Top panel, autoradiography of the 130-kDa bands on a typical Western blot of the postmitochondrial supernatants (80 µg protein/lane) with an antibody against mouse iNOS and visualization with a luminol reaction. Middle panel, Mean intensity of the respective bands determined by densitometry (n = 8). Values represent means ± SEM. Bottom panels, Comparison of the intensities for the 130-kDa iNOS band on gels corresponding to fractions from either the adult or the old animals to show reproducibility from rat to rat. See Results regarding the top band. C+, positive control (induced RPSMC). Different letters in superscripts denote significant differences between those groups (P < 0.01).

 


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  		Figure 4. Effect of aging on iNOS levels in the rat frontal cortex, cerebellum, and parietal cortex. Top panels, Autoradiography of the 130-kDa iNOS bands as on Fig. 3Go (cerebellum, 60 µg protein/lane; others, 30 µg/lane). Middle panels, Mean intensity of the respective bands determined by densitometry (N = 5). Values represent means ± SEM. Top band is not iNOS. Left, Frontal cortex; right, cerebellum. Bottom panel, Autoradiography of the 130-kDa band in the fractions obtained from the four brain regions from two old rats, with equal protein loads (50 µg) in all cases. C+, positive control (induced RPSMC); HYP, hypothalamus; CER, cerebellum; FCTX, frontal cortex; PCTX, parietal cortex. Different letters in superscripts denote significant differences between those groups (P < 0.05).

 
To validate the previous comparison between old and adult tissue, new immunoblots were conducted where all specimens from the adult hypothalamus were run on a single gel, concurrently with all specimens from the old hypothalamus run on another single gel. The intensity of iNOS expression was remarkably similar in the old hypothalamus from the 8 rats shown on Fig. 3Go (bottom), and virtually absent in the other series of samples from the adult hypothalamus run on a separate gel and developed simultaneously under identical conditions. The densitometric analysis of the gels presented on the top panel indicates (Fig. 3Go, middle panel) that iNOS expression is 3.8- and 7.6-fold higher in the old hypothalamus as compared with the adult and immature tissue, respectively.

To determine whether this aging-dependent iNOS expression is unique to the hypothalamus or is present in other sections of the brain, the Western blot analysis was repeated for fractions from the frontal cortex, cerebellum, and parietal cortex. Figure 4Go (top panel) shows that the pattern of iNOS expression among the age groups observed in the hypothalamus was reproduced in the frontal cortex (left panel) and cerebellum (right panel), as indicated by the 130-kDa band. The densitometric analysis (Fig. 4Go, middle) indicates the following respective increases in iNOS levels in old as compared with adult and immature rats:.3.9- and 12.8-fold (frontal cortex); 3.1- and 4.7-fold (cerebellum). The parietal cortex is not shown, but the densitometric values were 2.8- and 2.5-fold higher in the old rats than in the adult and immature animals, respectively, and the levels of expression were lower than in the other regions. Correcting for the different protein loads, the levels of iNOS expression in the old rat brain are hypothalamus>frontal cortex>cerebellum>>parietal cortex (Fig. 4Go, bottom).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This work is the first demonstration of an aging-related iNOS expression in the hypothalamus and other regions of the brain in experimental animals or humans. While NOS activity has been reported to be elevated in the brain from old rats (66, 67, 68, 69), regional differences, conflicting data (69, 70), and the lack of identification of the different NOS isoforms involved, make the interpretation difficult. Our results show that the excessive production of NO in the aged hypothalamus is not accompanied by a parallel increase in NMDAR number, refuting the hypothesis that NO synthesis is elevated in the postsynaptic neurons of old rats by the stimulation of nNOS activity through the NMDAR. Furthermore, the reduction of hypothalamic NMDAR with aging argues against a significant participation of these receptors in the increase in apoptosis and degenerative changes occurring in the brain of old rats (46, 71). This also confirms our previous work (8) showing that the GnRH efflux in incubations of fragments of preoptic area-medial basal hypothalamic in response to NMDA was considerably attenuated by aging.

The levels of iNOS protein detected in the hypothalamus and other brain regions of old rats are only moderate in comparison to those that can be generated in vitro or in vivo with iNOS inducers. This is consistent with the modest stimulation of NOS activity detected in the hypothalamic tissue cytosol. The activity measured by the L-arginine/citrulline conversion assay represents a balance between iNOS protein increase with aging and a putative partial down-regulation of nNOS activity, as a consequence of the reduction of NMDAR stimulation (16, 21) in the presence of constant nNOS protein content. The limited increase of hypothalamic overall NOS activity by aging probably explains why the 24-month-old Brown Norway rats do not exhibit the CNS manifestations associated with the massive cytotoxicity that would be observed when NO is released in situations such as septic shock, autoimmune processes, inflammatory conditions, or experimental administration of iNOS inducers to animals (23, 71). Specifically in the CNS, iNOS has been detected immunochemically in the brain of animals receiving LPS/interferon {gamma} (50, 51, 52, 53, 54, 55). The abnormal constitutive activation of iNOS to high levels by transcriptional regulation, as shown in glial cells and in neurons (23, 32, 72), as well as the relative Ca2+ independence of iNOS enzyme activity, are in contrast to the intermittent activation of nNOS activity by Ca2+ fluxes leading to physiological level of NO synthesis. Therefore, in the hypothalamus two separate pathways of NO synthesis catalyzed by at least two different NOS isoforms may exert paradoxical opposite effects on the same target, according to their respective cellular locations and the pattern of NO release.

It is likely that the spontaneous iNOS expression in the brain of old male rats, as compared with that occurring upon exogenous stimulation, would lead to a milder and gradual process of NO-induced neurotoxicity that may be either regional or generalized. In the hypothalamus, the effect may be general, including impairment of GnRH neuronal function. Aging has been shown in the rat to be accompanied by a reduction in the number of GnRH neurons in the medial preoptic area and the OVLT continuum in both male and female aged animals (11, 12, 13, 14), and a decrease of GnRH activation in aged female animals (15, 33). We have not excluded the possibility that the increased iNOS in the hypothalamus may affect other hypothalamic functions, such as regulation of GH secretion, energy homeostasis, thermogenesis, etc., which have shown to be altered with the aging process (73, 74, 75). In addition, NO synthesized by iNOS may also trigger feedback inhibition of nNOS activity, thus potentiating the enzyme down-regulation caused by NMDAR reduction (48).

The endogenous factors that induce iNOS expression in the aging brain are unknown, but by analogy with what is known on experimental iNOS induction, it can be postulated that this may occur through an increment in either the circulating levels or the local tissue release of cytokines. Recent reports show that TNF-{gamma} in the cerebrospinal fluid and peripheral circulation and IL-1ß and interferon-{gamma} are increased in monocytes by aging (44, 56, 57). Cytokines are also synthesized in situ in the hypothalamus (76), and the exogenous administration can block the nitroxidergic control of GnRH release both in vitro and in vivo (77) through iNOS induction.

Although the assumption that an excessive binding of EAA to NMDAR is at the root of many neurodegenerative disorders linked to aging, the few available studies measuring NMDAR number in different regions of the brain consistently showed a decrease with old age (34, 35, 36, 37, 38, 39). Our results in the hypothalamus not only extend these observations, but show that the reduction in EAA binding in vitro in this tissue is in fact due to a parallel decrease in the content of NMDAR protein. This is difficult to reconcile with the view that aging-associated damage in general and neuronal apoptosis in particular (22, 23, 58, 71, 78) in the CNS is due to the intensification of NMDAR-triggered signals, unless in vivo there is an increase in the level of EAA in contact with the target neurons that would compensate for the lower number of NMDARs. However, no significant changes have been observed in the circulating levels of EAA such as aspartate or glutamate with aging, and we have previously demonstrated (8) that the content of glutamate in the hypothalamus is in fact decreased by aging.

The physiological role of NMDARs in the positive control of GnRH pulsatile release through an NO-dependent pathway has been established by several groups (9, 10, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29). Therefore, because our results did not discriminate between NO synthesized from the NMDAR-modulated nNOS nitrergic neurons and NO synthesized by iNOS, we do not rule out the possibility that the decrease of NMDAR could in fact be an important factor in the alterations of GnRH release by aging. Similarly, it is necessary to determine whether the aging-related increase in iNOS expression and NOS activity does indeed correlate with an increase in the apoptotic index in hypothalamic GnRH neurons and a decrease in GnRH neuronal activity because the cytotoxic effects may be general and affect other hypothalamic neurons (e.g. GHRH and CRH neurons) and functions. The possibility of a widespread effect of iNOS spontaneous induction in other regions of the brain that are affected by neurodegenerative disorders associated with old age (79, 80, 81) needs to be ascertained. Should this be the case, the blockade of iNOS expression in the aging brain by targeted antisense gene therapy (29, 82) may become a therapeutic alternative worth exploring in experimental animals.


   Footnotes
 
1 This work was funded by grants from the NIH/NIDDK (DK07571), and from the Foundation for Research in Aging (FRA-97–1). Back

Received December 5, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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