Journal of Applied Physiology Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 100: 1983-1991, 2006. First published February 9, 2006; doi:10.1152/japplphysiol.01169.2005
8750-7587/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
100/6/1983    most recent
01169.2005v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, M.
Right arrow Articles by Galbraith, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, M.
Right arrow Articles by Galbraith, R. A.

The weight loss elicited by cobalt protoporphyrin is related to decreased activity of nitric oxide synthase in the hypothalamus

Muyao Li,1 Margaret A. Vizzard,2,3 Diane M. Jaworski,2 and Richard A. Galbraith3

Departments of 1Medicine, 2Anatomy and Neurobiology, and 3Neurology, University of Vermont, Burlington, Vermont

Submitted 15 September 2005 ; accepted in final form 3 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Administration of cobaltic protoporphyrin IX (CoPP) into the third ventricle of the brain by intracerebroventricular injection in rodents is known to result in transient hypophagia and remarkably prolonged weight loss. The mechanism of action of CoPP in eliciting these effects is unknown. It is known that nitric oxide plays a role in food intake and that the hyperphagia that results from a wide variety of genetic, physiological, and pharmacological stimuli can be blocked by the administration of inhibitors of the enzyme nitric oxide synthase (NOS). We demonstrate that intracerebroventricular administration of compounds that alter nitrergic tone can also change food ingestion and weight gain patterns in normophagic rats. We also demonstrate that CoPP decreases NOS activity but that it paradoxically increases neuronal NOS transcript expression and increases neuronal NOS protein content on Western blotting.

synthetic metalloporphyrin; nitric oxide activity; in situ hybridization; nitrergic tone; appetite

ADMINISTRATION OF THE SYNTHETIC metalloporphyrin cobaltic protoporphyrin IX (CoPP) to rodents is known to result in a dose-dependent, short-lived decrease in food intake and a prolonged reduction in body weight. Subcutaneous (sc) administration of CoPP (50 µmol/kg body wt) elicits reductions in appetite and body weight in rats, mice, chickens, and dogs (21, 23–25). Intracerebroventricular (icv) administration of CoPP leads to large reductions in body weight in normal and Zucker (fa/fa) rats, which can persist for over 300 days after a single injection of ~1% of the sc dose of CoPP (21, 22, 24). Reductions in food intake or body weight are also observed after intrahypothalamic injections of even smaller doses (approximately one-seventh the icv dose) into the paraventricular (PVN), dorsomedial, and ventromedial nuclei but not into the lateral area of the hypothalamus or the thalamus (26). It is also known that icv injection of the major orexigenic neuropeptide Y (NPY) is without effect in CoPP-treated rats, even though CoPP-treated rats with resultant hypophagia display appropriate compensatory increases in hypothalamic mRNA for NPY and unchanged numbers and affinity of hypothalamic NPY receptors (12, 69). Other investigators have shown a similar failure to feed in response to galanin and epinephrine in CoPP-treated animals (12). The mechanism of action of CoPP is unknown, but these observations suggest that its locus of action may be distal to the level of ligand-receptor interactions of at least some of the molecules that regulate feeding behavior in the central nervous system (CNS).

Nitric oxide (NO), in addition to its role in endothelial cells (47) and macrophages (42), has been shown to function as a neurotransmitter and a neuromodulator (7, 27). NO synthase (NOS) catalyzes the conversion of arginine into citrulline and NO. In the brain, neuronal NOS (nNOS) is a soluble enzyme that is dependent on NADPH, calcium, calmodulin, tetrahydrobiopterin, FAD, flavin mononucleotide, phosphorylation sites, and heme (6, 8, 37, 45). The highest activity of NOS has been reported to be in the cytosolic fraction of the cerebellum, but activity similar to that of whole brain was found in the hypothalamus (18). NOS has been mapped in the rat brain as NADPH-diaphorase (31), as nNOS immunoreactivity (34), and as nNOS using in situ hybridization (30). NO has been implicated, via activation of guanylate cyclase, to be the transducing agent in glutamate-induced N-methyl-D-aspartate receptor activation (27, 37) and in memory and long-term potentiation (11, 27, 61). The presence of NOS has been firmly established in both the rat (18, 53, 70) and human (4) hypothalamus, and its activity and product, NO, have been implicated in hypothalamic responses to cytokines (59), sexual behavior (41), actions of angiotensin II in the PVN (3), and in release from the hypothalamus of oxytoxin (67), vasopressin (73), prostaglandin E2 (56), corticotropin-releasing factor (33), somatostatin (73), and luteinizing hormone-releasing hormone (55, 57).

In mice, sc administration of L-arginine, the substrate for NOS, increased food intake, whereas competitive inhibitors of NOS [N{omega}-nitro-L-arginine (L-NNA) and its methyl ester L-NAME] produced an L-arginine-reversible decrease in food intake (49, 50). L-NAME also blocked the feeding effect of exogenous NPY, and repetitive administration of the inhibitor over 5 days caused weight loss (50), whereas L-NNA induced anorexia in obese Zucker rats (66). NO has been reported to control feeding behavior in chickens (13) and even exerts this function as far back in the phylogenetic tree as Hydra vulgaris, the most primitive invertebrate with a nervous system (14). In rodents, the hyperphagia attributed to 2-deoxyglucose (72), chlordiazepoxide (15), 8-hydroxy-2-di-n-(propylamino)tetralin (71), highly palatable milk (48), NPY (50), homozygosity for the ob gene (51), homozygosity for the fa gene (63, 66), food deprivation (64), morphine (10), and stress (16) has, in each case, been shown to be abrogated by the administration of inhibitors of NOS. In almost every case, the blockade of NOS was reported to be reversible by L- but not D-arginine. In the case of food deprivation and homozygosity for the ob gene, NOS activity was increased, respectively, in the diencephalon (64) and specifically in the hypothalamus (52). The above list of stimuli for hyperphagia involves a wide array of known and unknown signal transduction systems. It seems unlikely that NO would act separately before or at each individual receptor in each specific region of the hypothalamus or brain. A more likely hypothesis (presented in Table 1) is that NOS acts downstream of these receptors in some common pathway, which leads ultimately to the behavior of increased food ingestion.


View this table:
[in this window]
[in a new window]
  		Table 1. Model for nitric oxide-dependent hyperphagia

 
In view of the parallelism of these studies involving NO and CoPP, we hypothesized that CoPP may act, at least in part, by modifying nitrergic tone within the CNS. This paper describes a series of experiments to examine this hypothesis in adult Sprague-Dawley rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
CoPP was purchased from Porphyrin Products (Logan, UT). All other chemicals were of the highest reagent grade commercially available.

Animal handling and treatment.   These protocols and all animal handling were approved by and done in accordance with the guidelines and requirements of the University of Vermont Institutional Animal Care and Use Committee. Adult male Sprague-Dawley rats (mycoplasma free; 200 g) were purchased from Charles River (Quebec, Canada) and housed in an air-conditioned (23 ± 1°C) room with lights on for 12 h daily (starting at 6:00 AM) in the University of Vermont Animal Facility. Rats were housed singly in regular cages with free access to Purina Rat Chow (RMH 3000) and water for at least 5 days before surgery. Stereotactic surgery was used to implant chronic indwelling stainless steel cannulas into the third ventricle of the brain under fentanyl-droperidol anesthesia. Coordinates for cannula placement were obtained from the Konig Klippel stereotactic surgery atlas (38); with the nose bar 2.4 mm below the intra-aural line and the vertical micrometer gantry tilted 9° toward the animal, the coordinates were 4.9 mm dorsal to the intra-aural line, 5 mm anterior to the intra-aural line, and 1.1 mm lateral to the midline (24). A minimum of 5 days later, animals were injected icv with CoPP (which was dissolved in 0.2 N NaOH and the pH adjusted to 7.4–7.8) or other experimental compounds. Control animals received equal volumes of vehicle. Volumes of icv injections never exceeded 10 µl per animal and were administered over 5–10 s. In some experiments, animals received two or more icv injections on different days. Measures of body weight were made daily with a digital balance with a 6-s integration period to minimize the effects of animal movement. For experiments involving measurement of food intake, animals were housed after stereotactic surgery in individual Nalgene metabolic cages and given additional time for acclimation to the cage and feeding system. Food intakes were measured using powdered rat chow with daily or hourly weigh-backs as indicated.

Real-time PCR.   Total RNA was isolated from rat hypothalamic blocks with the Ultraspec-II RNA Isolation System (Biotecx Laboratories, Houston, TX). The first-strand cDNA was synthesized from 2 µg of RNA in a 20-µl reverse transcription reaction mixture containing Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Tech, Chicago, IL). Expression of nNOS was quantified by TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA). The customer-designed Assays-on-Demand Gene Expression primers and MGB probe (FAM dye-labeled) for rat nNOS were as follows: forward primer, 5'-CACCAGCACCTTTGGCAATG-3'; reverse primer, 5'-GGTGCCTCATCTCCATTAAAGCA-3'; and TaqMan probe, 5'-CAGCCGAATTTCTCC-3'. For inducible NOS (iNOS), the TaqMan gene expression assay Rn00561646-ml was used. Rat GAPDH proprietary primers (Assay ID Rn01462661_g1; Applied Biosystems) were used. The real-time PCR was performed by the Vermont Cancer Center and the University of Vermont DNA facility. One microliter of each cDNA sample was mixed with 24 µl of Master Mix in a 96-well TaqMan plate and amplified using an ABI Prism 7700 instrument at 50°C, 2 min; 95°C, 10 min; then followed by 95°C, 15 s; and 60°C, 1 min for 40 cycles. The data were analyzed using software supplied with the ABI Prism 7700.

In situ hybridization.   At least three rats per group were used for each probe. Brains were dissected as previously described (24) and were cut vertically to remove the anterior portion at the optic chiasm and the posterior portion at the interpeduncular fossa so that the remaining blocks included the whole hypothalamus with a small additional margin of adjacent tissue. The blocks were immediately frozen on foil on dry ice, then stored at –80°C. Blocks were sectioned in a cryostat (Micron HM500O) at –20°C and at a thickness of 13 µm. In situ hybridization was performed as previously described (32).

cDNAs were prepared as follows: 1) nNOS, GenBank no. U67309, nt 2561–2845; and 2) heme oxygenase 1 (HO1), GenBank NM-012580, nt 4676–4930. Radiolabeled cDNA probes were generated using T7 or SPG RNA polymerase in the PCRII-TOPO vector (Invitrogen) in an in vitro transcription mixture that consisted of 1 µl x10 buffer; 0.5 µl each of 10 mM solutions of rATP, rGTP, and rUTP; 0.6 µl 0.2 mM rCTP; 20 units Rnasin RNase inhibitor (Promega); 1.5 µg DNA template; 3 µl [35S]-5'{alpha}-CTP; and 50 units T7 or SP6 polymerase (New England Biolabs, Beverly, MA) at 37°C for 1 h. The probes then underwent DNase digestion to remove remaining template and a phenol-chloroform purification to remove DNase. The counts per minute (cpm) value was determined by counting in a liquid scintillation spectrometer.

After prehybridization and hybridization, the slides were dipped in NTB-2 emulsion (Kodak) and exposed at 4°C for ~3 wk. The slides were developed in Kodak D19 developer for 5 min, fixed in Kodak Fixer for 4 min, dehydrated through graded ethanols, and coverslipped with Permount. Slides were observed under dark-field microscopy (Nikon, Tokyo, Japan), and silver grains in the region of the hypothalamus were assessed. Images were captured at x10 magnification with SPOT image software (Diagnostics Instrument, Sterling Heights, MI) and processed using Photoshop. Regions of interest (e.g., specific hypothalamic nuclei) were outlined by electronic pen, and the area thus enclosed was cropped and stored as a TIFF file. Photoshop was used to quantitate the silver grains by comparing the white pixel count to the total pixel count of the cropped area of interest. A minimum of three images, when possible, that corresponded to the anterior, middle, and posterior sections of the region of interest were analyzed from each rat studied.

Western immunoblotting experiments.   Total protein was extracted from rat hypothalamic blocks, as described previously (24). Protein samples (15 µg) were electrophoresed on 7.5% SDS-PAGE gels and then electroblotted onto 0.2-µm nitrocellulose membranes. After blocking of membranes with 1% BSA in PBS Tween buffer, the membranes were incubated with the rabbit polyclonal antibody for nNOS (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) overnight, shaking at 4°C. After washing, membranes were incubated with secondary species-specific antibodies (1:1,000). The proteins were visualized using a SuperSignal West Pico Trial Kit (Pierce Biotechnology, Rockford, IL) and exposed to radiographic films. The membranes were stripped and then reimmunoblotted with anti-beta-actin as a loading control (Abcam, Cambridge, MA). Using Quantity One Software (Bio-Rad, Hercules, CA), the density of the nNOS bands was divided by the densities of each respective beta-actin band to yield relative units.

Immunohistochemistry experiments.   In experiments involving immunohistochemistry, animals were injected icv with 0.4 µmol/kg body wt CoPP or with vehicle. Four hours after injection, animalswere anesthetized with halothane and perfused through the left ventricle of the heart, initially with oxygenated Krebs buffer and then with ~350 ml of 4% paraformaldehyde in 0.1 M NaCl, pH 7.4, PBS. Brains were extracted from the skull and hypothalamic blocks were dissected [by coronal cuts anterior and posterior to hypothalamic landmarks (24)] and postfixed in 4% paraformaldehyde solution at 4°C overnight. The following day, the blocks were transferred to 30% sucrose and incubated overnight at 4°C. After freezing in optimum cutting temperature compound (Sakura, Tokyo, Japan), blocks were sectioned coronally at 40 µm on a cryostat, and sections were individually floated in multiwell dishes filled with PBS. Sections were treated for 30 min with 0.4% Triton detergent in potassium PBS to aid in antibody penetration. Primary antibodies were added, and the plates were shaken gently for 1 h at room temperature and incubated at 4°C for 72 h. Sections were then brought to room temperature while shaking, and the primary antibody solution was removed by washing three times with PBS buffer. After removal of the last wash, the secondary antibody solution was added, and the plates were incubated for 2 h at room temperature in the dark. Removal of the second antibody was achieved by washing three times with PBS buffer before mounting on subbed slides. Tissues were allowed to dry, placed in PBS buffer, and coverslipped with Citifluor (Ted Pella, Reading, CA).

Fos studies were performed using indirect fluorescence visualization. The method utilized rabbit c-Fos-AB5 (1:2,000; Oncogene Research Products, Cambridge, MA) with goat anti-rabbit Cy3 (1:500; Jackson Immuno Research Laboratories, West Grove, PA) as the secondary antibody. Double labeling experiments to identify colocalization of Fos and nNOS utilized anti-nNOS antibody (1:3,000; Santa Cruz Biotechnology). Species-specific secondary antibodies (Cy2 or FITC-conjugated) were used for colocalization experiments. Sections were examined using a fluorescence microscope, and digital images were captured with Magnafire 2.1 software and imported into Photoshop for printing. Tissue incubated in the absence of primary or secondary antisera showed no reaction products. Staining observed in experimental tissue was compared with that observed from experiment-matched negative controls. Nuclei exhibiting immunoreactivity that was greater than the background level observed in experiment-matched negative controls were considered positively stained. Positively stained nuclei were not further divided into categories of different staining intensities.

NO synthesis activity.   NOS activity was measured in hypothalamic blocks, which were rapidly dissected, washed free of blood, and snap-frozen in liquid nitrogen. The principle of the assay is the NOS-dependent conversion of radiolabeled arginine to radiolabeled citrulline, which is separated using anion-exchange column chromatography. Briefly, and as described (29), homogenates were incubated, shaking, in 100 mM HEPES buffer, pH 7.5, with 50 µM tetrahydrobiopoterin, 1 mM CaCl2, 4 µM FAD, 1 µM calmodulin, and 200 µM [6-14C]arginine for 15 min at 37°C; the reaction was initiated by the addition of 100 µM NADPH and quenched by the addition of trichloroacetic acid. After neutralization with 1.5 M HEPES buffer, aliquots were applied to disposable columns containing Dowex X-8 in the sodium form and eluted with water. After scintillation counting, results were corrected for the radioactivity in identical incubations carried out without NADPH. Experiments to determine the direct effect of addition of CoPP to the assay were carried out on pooled hypothalamic homogenates from untreated animals to which CoPP or vehicle were added before incubation in the assay. Protein content of the homogenates was measured by the method of Lowry et al. (40).

Statistics.   Food intakes after injections of NOS inhibitors were normalized to percentage of respective baseline food intakes and analyzed utilizing Student's t-test. NOS activities are reported as disintegrations per minute per milligram protein, and differences in means were analyzed utilizing Student's t-test, or by ANOVA and Dunnett's test in experiments involving direct addition of CoPP to the assay.

Pixel counts from outlined hypothalamic nuclei in in situ hybridization experiments were combined to form a mean per animal (minimum of 3 sections). The means from each animal for each specific nucleus were combined to form a means per nucleus per treatment group, which were analyzed nonparametrically by the Wilcoxon two-sample test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Studies with NO inhibitors.   The icv administration of CoPP is well established to cause a transient decrease in food intake and a prolonged reduction in body weight. The first experiments were, therefore, designed to determine whether factors known to modify nitrergic tone in other circumstances elicited effects similar to the known effects of CoPP on appetite and body weight when administered via the same route (icv) into the brain of normal rats. L-NNA was utilized because it has been reported to selectively and potently inhibit the nNOS found in the CNS (17, 19, 39). As shown in Fig. 1, animals continued their gradual weight gain after initial control icv injections of saline on day 1 but showed progressive weight loss following subsequent icv injections of L-NNA (100 µg/rat) on days 3 and 7. In contrast, rats injected with a less nNOS-specific inhibitor, L-NAME (300 µg/rat), with the same dosing frequency, gained weight steadily throughout the experiment. After the saline injection and after each of the L-NNA or L-NAME injections, 24-h food intakes of rats were measured. Results are shown in Table 2. It is clear from these data that the weight loss seen after icv administration of L-NNA is accompanied by decreases in food intake, whereas, in rats treated with L-NAME, there was no diminution in food intake commensurate with the lack of an effect on weight gain.


Figure 1
View larger version (9K):
[in this window]
[in a new window]
  		Fig. 1. The effects of intracerebroventricular (icv) administration of nitric oxide synthase (NOS) inhibitors on weight gain in rats. Adult male rats were implanted with stainless steel cannulas in the third ventricle of the brain and acclimated to individual housing in metabolic cages, as described in MATERIALS AND METHODS. On day 1, all animals received saline (vehicle) icv. On days 3 and 7, animals were re-injected icv with N{omega}-nitro-L-arginine 100 µg/rat ({blacksquare}) or N{omega}-nitro-L-arginine methyl ester 300 µg/rat ({circ}). Animals were exposed to water and powdered food ad libitum. Mean weights ± SE are presented for four rats per group.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Effect on NOS inhibitors on food intakes

 
NOS activity.   To determine whether the weight loss caused by CoPP was also related to inhibition of NOS activity, as with the NOS inhibitor nitroarginine, NOS activity was measured in CoPP-treated rats. Animals treated icv with saline gained ~20 g over the subsequent 48 h, whereas those treated with CoPP (0.4 µmol/kg body wt) lost ~20 g over the same period. NOS activity in hypothalamic blocks from these animals was determined to be significantly reduced, by ~20% (P < 0.01), in CoPP-treated rats compared with saline-treated rats (see Table 3). To determine whether or not CoPP inhibited NOS activity via a direct effect, either vehicle or different concentrations of a CoPP solution were added to hypothalamic homogenates from untreated rats. As can be seen in Fig. 2, there was a highly significant (P < 0.001) dose-dependent inhibition of NOS activity caused by direct addition of CoPP.


View this table:
[in this window]
[in a new window]
  		Table 3. Effect of intracerebroventricular CoPP on nitric oxide synthase activity in the hypothalamus

 

Figure 2
View larger version (6K):
[in this window]
[in a new window]
  		Fig. 2. Direct addition of Co3+ protoporphyrin IX (CoPP) inhibits NOS activity. Hypothalami were harvested and pooled from untreated rats. After homogenization, vehicle or CoPP, at the indicated dosages, was added directly to the homogenates. These homogenates were then assayed for NOS activity, as described in MATERIALS AND METHODS, within 15 min of addition of vehicle or CoPP. CPM, counts per minute. *P < 0.0001 vs. control and vs. each other (Dunnett's).

 
nNOS mRNA concentrations.   The concentration of NOS transcripts was quantitatively examined, using reverse transcription and real-time polymerase chain reaction, in vehicle- and CoPP-treated rat hypothalami. As shown in Table 4, both nNOS and iNOS expression in the hypothalamus were increased about threefold following treatment with CoPP (P = 0.0067 and P = 0.041, respectively).


View this table:
[in this window]
[in a new window]
  		Table 4. Effect of CoPP on hypothalamic nNOS and iNOS mRNA concentrations

 
Experiments were also performed to determine the effect of CoPP on the distribution of nNOS gene expression using in situ hybridization. Representative photomicrographs are shown in Fig. 3. Results of analysis of these in situ hybridization studies are presented in Table 5. There was no significantly different distribution of nNOS mRNA in brains of animals treated with CoPP compared with vehicle. There was a trend of an increase in the posterior hypothalamic area with CoPP, but it did not achieve statistical significance. In contrast, and consistent with the known induction of HO1 elicited by treatment with CoPP (24), the hybridization signal for the positive control, HO1, was greatly increased by CoPP administration compared with the negative results following administration of vehicle (Fig. 4) and indicates the integrity of the technique.


Figure 3
View larger version (74K):
[in this window]
[in a new window]
  		Fig. 3. The effects of icv CoPP administration on in situ hybridization of neuronal NOS (nNOS) mRNA in rat brains. Rats were treated icv with vehicle (n = 3) or CoPP 0.4 µmol/kg body wt (n = 3). After 48 h, animals were killed, and brains were harvested, sectioned, and used for in situ hybridization with nNOS probes as described in MATERIALS AND METHODS. A, C, and E: hybridization patterns from CoPP-treated antisense, saline-treated antisense, and saline-treated sense probes, respectively, in sections taken at –6.2 mm from the intra-aural line. B, D, and F: the same sections as in A, C, and E but after restaining with 0.5% cresyl violet and examination under the light microscope to identify tissue architecture. The calibration bar in F represents 250 µm. DM, dorsomedial nucleus; VM, ventromedial nucleus; A, arcuate nucleus.

 

View this table:
[in this window]
[in a new window]
  		Table 5. Effect of CoPP on in situ hybridization of nNOS mRNA in rat brain

 

Figure 4
View larger version (76K):
[in this window]
[in a new window]
  		Fig. 4. The effects of icv CoPP administration on in situ hybridization of heme oxygenase 1 (HO1) mRNA in rat brains. Sections from the brains of the same animals described in the legend to Fig. 3 were used for in situ hybridization with HO1 probes as described in MATERIALS AND METHODS. A: typical silver grain hybridization pattern from a CoPP-treated animal; B: that from a vehicle-treated control animal. The spatial coordinates of the sections are essentially identical to those of the sections displayed in Fig. 3. The calibration bar in B represents 250 µm.

 
nNOS protein concentrations.   Further experiments were carried out to determine whether the reduced NOS activity was related to a decrease in the concentration of nNOS protein. As shown in Table 6, 48 h after icv treatment with CoPP 0.4 µmol/kg body wt, animals lost, on average, ~10% of their body weight compared with an ~3% increase in the weights of control rats. This weight loss was accompanied by an ~50% increase in the concentration of nNOS protein, as assessed by Western blotting (Table 6 and Fig. 5).


View this table:
[in this window]
[in a new window]
  		Table 6. Effect of CoPP on hypothalamic nNOS immunoreactivity

 

Figure 5
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 5. Western blots of nNOS in the hypothalamus. Rats were injected icv with saline or CoPP 0.4 µmol/kg body wt, and 48 h later, hypothalami were harvested and processed for Western blot analysis as described in MATERIALS AND METHODS. Representative blots from one experiment are shown. The densitometry data, normalized to beta-actin band densities, are presented in Table 6.

 
nNOS immunohistochemistry.   Indirect immunofluorescence was utilized to determine whether the increased expression of c-fos known to occur following administration of CoPP (20) was colocalized with nNOS immunoreactivity in the brain. Analysis of the entire brain section generally and the hypothalamus specifically showed no evidence of any colocalization of Fos immunoreactivity with nNOS immunoreactivity. Although nNOS and Fos were clearly present in the same or adjacent areas, specifically in the hypothalamus at the base of the third ventricle (Fig. 6) and generally in extrahypothalmic sites such as the reticular thalamic nucleus (data not shown), there was no evidence to support colocalization of these proteins in the same cells.


Figure 6
View larger version (33K):
[in this window]
[in a new window]
  		Fig. 6. Dual labeling of nNOS and Fos in the hypothalamus of CoPP-treated rats. Adult male rats were treated icv with CoPP 0.4 µmol/kg body wt. Four hours later, brains were perfused and processed for immunohistochemistry as described in MATERIALS AND METHODS. A: representative photomicrograph (x20). Fos immunoreactivity (IR) (red staining) and nNOS IR (green staining) are both clearly visible near the base of the third ventricle. However, the majority of Fos staining is in the arcuate (Arc) nucleus, whereas the majority of nNOS staining is in the median eminence (ME). There was no colocalization. The calibration bar represents 80 µm. B: the location from which the photomicrograph was taken. D3V, dorsal third ventricle; 3V, third ventricle.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Multiple lines of evidence (see Table 1) suggest that NO contributes to the regulation of food intake in response to a wide range of physiological, genetic, and pharmacological triggers. CoPP is a synthetic metalloporphyrin, which is known to elicit hypophagia and weight loss in a number of different species (21, 23–26), including animals genetically programmed to become obese (22). CoPP is known from icv treatments to act in the CNS, to reside within the hypothalamus for >5 wk, and to activate synthesis of Fos in the hypothalamus and ventricular system (20). However, its precise mechanism of action remains elusive. The goal of the experiments described was, therefore, to determine whether or not NO plays a role in CoPP-induced alterations in food intake.

The first experiments were designed to confirm that agents known to perturb nitrergic tone in other situations were active in the CNS in altering food intake in other than obese or hyperphagic rats. Administration of the more nNOS-specific inhibitor L-NNA was effective in reducing both food intake and weight (Fig. 1 and Table 2), whereas the less specific inhibitor L-NAME was without effect. The examples of hyperphagic stimulants blocked by NOS inhibitors in Table 1 establish that NOS inhibitors are active in modifying food intake under conditions that drive hyperphagia. However, our observations of the effects of L-NNA on food intake described above extend this notion to suggest that NOS inhibitors can also modify food intake, not only under conditions that elicit hyperphagia, but also under normophagic circumstances. The pharmacological agent was administered icv into the dorsal third ventricle of the brain; hence the objective of demonstrating that agents known to perturb nitrergic tone in other circumstances could alter food intake after direct injection into the brain in normal animals was met.

We demonstrated that there was an ~20% decrease in the activity of NOS in hypothalamic tissues 48 h after icv treatment with CoPP. Furthermore, this inhibition can also result from a direct action of CoPP added to the NOS assay. Clearly, the method used to measure NOS activity does not allow for differentiation between the concentrations of iNOS, endothelial NOS (eNOS), and nNOS. However, the number of observations delineating the existence and role of nNOS in the hypothalamus (3, 4, 18, 33, 41, 53, 55–57, 59, 67, 70, 73) suggests that the majority of this measured NOS activity likely derives from nNOS activity.

Next, we examined the effect of icv treatment with CoPP on gene expression in hypothalami. Although we have previously detected both nNOS and iNOS in the hypothalamus by using RT-PCR (data not shown), in view of the problems of quantitation from ordinary RT-PCR reactions, real-time PCR was used to measure nNOS expression. Although there was clearly an increase in nNOS and iNOS expression elicited by treatment with CoPP, analysis of in situ hybridization studies failed to reveal differences in the distribution of hybridization to nNOS probes in any hypothalamic or extrahypothalamic site (Fig. 2 and Table 5) or in hybridization to iNOS probes (data not shown). On the other hand, there was a clear and large increase in HO1 expression in in situ hybridization studies with CoPP. This inducing effect of CoPP on HO1, although well documented, is unlikely to play a role in the alterations in food intake and body weight typical of CoPP. The evidence for this is that coadministration of CoPP with tin-protoporphyrin, a known inhibitor of HO activity, normalized HO activity but had no effect on the decreased appetite and weight loss elicited by CoPP (24).

The regulation of nNOS, as indicated by its original name, constitutive NOS, has long been held to be catalytic rather than transcriptional. Indeed, there exists a plethora of modulators of nNOS catalytic activities, including NADPH, calcium, calmodulin, tetrahydrobiopterin, FAD, flavin mononucleotide, phosphorylation sites, and feedback binding of its product, NO, to its constituent heme molecule (1, 2, 6, 8, 28, 36, 37, 45, 62). It is known that various metal-substituted protoporphyrins can be assembled into the heme binding pocket of myolobin (68), hemoglobin (74), cytochrome b5 (54), and cytochrome P-450 (5, 46). It has also been shown that CoPP can reconstitute nNOS from apo-nNOS in vitro; that it is less effective than iron-, tin-, zinc-, or manganese-protoporphyrin in this assembly process; and that none of the iron-substituteed protoporphyrins supports the synthesis of NO in the presence of NADPH and L-arginine (60). Although the heme of nNOS can be disassociated by the mechanism-based inactivator aminoguanidine (9), it is not known if the heme contained in nNOS is exchangable with CoPP in vivo. Clearly, CoPP acts in some manner to reduce NOS activity, but the mechanism of its interaction with the enzyme is unknown.

The approximately threefold increase in iNOS and nNOS mRNA concentrations and the 60% increase in NOS protein, in the face of an ~20% decrease in NOS activity, was surprising. It is possible that the majority of the reduction in activity resulted from inhibition of the activity of eNOS and/or iNOS proteins; however, as described earlier, this seems unlikely. Despite the existing dogma that nNOS is only regulated catalytically, it is known from other studies that not only the activity (65) but also immunoreactivity (35) of nNOS in the PVN is increased by fasting. If such increases were a normal homeostatic mechanism in response to fasting, then the "fasting" (i.e., decreased food intake) caused by administration of CoPP might lead to similar compensatory increases in nNOS mRNA expression and immunoreactive nNOS. Indeed, in the case of CoPP, it may be that the stimulus to increase nNOS expression may be the reduction in NOS activity caused by CoPP. Alternatively, it is possible that the decrease in NOS activity and the resulting decrease in the concentration of NO leads to a decrease in S-nitrosylation. It is known that the transcriptional factor NK-{kappa}B can be inhibited by S-nitrosylation (44), by repression of the inhibitory NF-{kappa}B kinase (58). Thus S-nitrosylation-induced derepression of NF-{kappa}B may lead, as is known to occur (43), to upregulated expression of iNOS. A similar regulatory mechanism may pertain to nNOS and might account for the increased expression of nNOS mRNA and protein in the face of continued inhibition of the catalytic activity of both preexisting and newly synthesized nNOS protein. Such inhibition of newly synthesized nNOS protein is consistent with the observation that 57CoPP can be detected in the hypothalamus for at least 5 wk after a single icv injection (20). Further support for this concept is the absence of colocalization of nNOS with c-fos immunoreactivity in the hypothalamus (Fig. 6), suggesting that the changes in iNOS transcription did not result from a direct activating effect of CoPP (as evidenced by c-fos expression). This observation is more consonant with a secondary regulatory action following the inhibition of NOS activity caused by CoPP.

In summary, we have shown that icv administration of modulators of nitrergic tone influence feeding behavior and weight gain in normal rats. The hypophagia and weight loss observed after icv administration of CoPP are accompanied by decreases in NOS activity, increases in nNOS mRNA expression, and an increase in nNOS immunoreactivity in the hypothalamus. Further studies will need to assess the mechanisms by which CoPP acts to reduce the enzyme activity of NOS in the hypothalamus yet induces nNOS transcription and translation. Such studies may not only add to our understanding of appetite and weight homeostasis but may also point the way to pharmacological interventions.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-53479 to R. A. Galbraith. The use of TaqMan was supported by the Vermont Cancer Center's National Institutes of Health Grant P30 CA-22435 to D. Yandell.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Galbraith, Dept. of Medicine, Univ. of Vermont, C209 Given Bldg., 89 Beaumont Ave., Burlington, Vermont 05405 (e-mail: richard.galbraith{at}uvm.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abu-Soud HM, Feldman PL, Clark P, and Stuehr DJ. Electron transfer in the nitric-oxide synthases. J Biol Chem 269: 32318–32326, 1994.[Abstract/Free Full Text]
  2. Abu-Soud HM, Yoho LL, and Stuehr DJ. Calmodulin controls neuronal nitric-oxide synthase by a dual mechanism. J Biol Chem 269: 32047–32050, 1994.[Abstract/Free Full Text]
  3. Bains JS and Ferguson AV. Angiotensin II neurotransmitter actions in paraventricular nucleus are potentiated by a nitric oxide synthase inhibitor. Regul Pept 50: 53–59, 1994.[Web of Science][Medline]
  4. Bernstein HG, Stanarius A, Baumann B, Henning H, Krell D, Danos P, Falkai P, and Bogerts B. Nitric oxide synthase-containing neurons in the human hypothalamus: reduced number of immunoreactive cells in the paraventricular nucleus of depressive patients and schizophrenics. Neuroscience 83: 867–875, 1998.[CrossRef][Web of Science][Medline]
  5. Bornheim LM, Correia MA, and Smith KM. Functional reconstitution of rat liver cytochrome P-450 with mesohemin. Biochem Biophys Res Commun 121: 95–101, 1984.[CrossRef][Web of Science][Medline]
  6. Bredt DS, Hwang PM, Glatt CE, Lowenstein C, Reed RR, and Snyder SH. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 351: 714–718, 1991.[CrossRef][Medline]
  7. Bredt DS and Snyder SH. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc Natl Acad Sci USA 86: 9030–9033, 1989.[Abstract/Free Full Text]
  8. Bredt DS and Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 87: 682–685, 1990.[Abstract/Free Full Text]
  9. Bryk R and Wolff DJ. Pharmacological modulation of nitric oxide synthesis by mechanism-based inactivators and related inhibitors. Pharmacol Ther 84: 157–178, 1999.[CrossRef][Web of Science][Medline]
  10. Calignano A, Persico P, Mancuso F, and Sorrentino L. Endogenous nitric oxide modulates morphine-induced changes in locomotion and food intake in mice. Eur J Pharmacol 231: 415–419, 1993.[CrossRef][Web of Science][Medline]
  11. Chapman PF, Atkins CM, Allen MT, Haley JE, and Steinmetz JE. Inhibition of nitric oxide synthesis impairs two different forms of learning. Neuroreport 3: 567–570, 1992.[Web of Science][Medline]
  12. Choi SJ, Meeran K, O'Shea D, Lambert PD, and Bloom SR. Weight loss in rats treated with intracerebroventricular cobalt protoporphyrin is not specific to the neuropeptide Y system. Brain Res 729: 223–227, 1996.[Web of Science][Medline]
  13. Choi YH, Furuse M, Okumura J, and Denbow DM. Nitric oxide controls feeding behavior in the chicken. Brain Res 654: 163–166, 1994.[CrossRef][Web of Science][Medline]
  14. Colasanti M, Lauro GM, and Venturini G. NO in hydra feeding response. Nature 374: 505, 1995.[Medline]
  15. Czech DA. Possible involvement of nitric oxide in chlordizepoxide-induced feeding in the mouse. Pharmacol Biochem Behav 55: 327–331, 1996.[CrossRef][Web of Science][Medline]
  16. Czech DA, Klosterman AE, and Le Sueur KT. NG-nitro-L-arginine methyl ester reduces stress-related feeding in the rat tail-pinch model. Pharmacol Biochem Behav 60: 91–96, 1998.[CrossRef][Web of Science][Medline]
  17. Dwyer MA, Bredt DS, and Snyder SH. Nitric oxide synthase: irreversible inhibition by L-NG-nitroarginine in brain in vitro and in vivo. Biochem Biophys Res Commun 176: 1136–1141, 1991.[CrossRef][Web of Science][Medline]
  18. Forstermann U, Gorsky LD, Pollock JS, Schmidt HW, Heller M, and Murad F. Regional distribution of EDRF/NO-synthesizing enzyme(s) in rat brain. Biochem Biophys Res Commun 168: 727–732, 1990.[CrossRef][Web of Science][Medline]
  19. Furfine ES, Harmon MF, Paith JE, and Garvey EP. Selective inhibition of constitutive nitric oxide synthase by L-NG-nitroarginine. Biochemistry 32: 8512–8517, 1993.[CrossRef][Medline]
  20. Galbraith RA, Hodgdon I, Grimm MS, and Vizzard MA. Prolonged retention of the anorectic cobalt protoporphyrin in the hypothalamus and the resulting expression of Fos. Am J Physiol Regul Integr Comp Physiol 287: R465–R471, 2004.[Abstract/Free Full Text]
  21. Galbraith RA and Kappas A. Regulation of food intake and body weight by cobalt porphyrins in animals. Proc Natl Acad Sci USA 86: 7653–7657, 1989.[Abstract/Free Full Text]
  22. Galbraith RA and Kappas A. Cobalt-protoporphyrin suppresses expression of genetic obesity in homozygous (fa/fa) Zucker rats. Pharmacology 41: 292–298, 1990.[CrossRef][Web of Science][Medline]
  23. Galbraith RA and Kappas A. Cobalt protoporphyrin regulates body weight in beagle dogs. Induction of weight loss in normal animals of stable adult weight. Pharmacology 43: 96–105, 1991.[Web of Science][Medline]
  24. Galbraith RA and Kappas A. Intracerebroventricular administration of cobalt protoporphyrin elicits prolonged weight reduction in rats. Am J Physiol Regul Integr Comp Physiol 261: R1395–R1401, 1991.[Abstract/Free Full Text]
  25. Galbraith RA and Kappas A. Regulation of food intake and body weight in rats by the synthetic heme analogue cobalt protoporphyrin. Am J Physiol Regul Integr Comp Physiol 261: R1388–R1394, 1991.[Abstract/Free Full Text]
  26. Galbraith RA, Kow LM, Pfaff D, and Kappas A. Injection of cobalt protoporphyrin into the medial nuclei of the hypothalamus elicits weight loss. Am J Physiol Regul Integr Comp Physiol 263: R805–R812, 1992.[Abstract/Free Full Text]
  27. Garthwaite J, Charles SJ, and Chess-Williams R. Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intracellular messenger in the brain. Nature 336: 385–388, 1988.[CrossRef][Medline]
  28. Griscavage JM, Fukuto JM, Komori Y, and Ignarro LJ. Nitric oxide inhibits neuronal nitric oxide synthase by interacting with the heme prosthetic group. J Biol Chem 269: 21644–21649, 1994.[Abstract/Free Full Text]
  29. Hevel JM and Marletta MA. Nitric-oxide synthase assays. Methods Enzymol 233: 250–258, 1994.[Web of Science][Medline]
  30. Iwase K, Iyama K, Akagi k Yano S, Fukunaga K, Miyamoto E, Mori M, and Takiguchi M. Precise distribution of neuronal nitric oxide synthase mRNA in the rat brain revealed by non-radioisotopic in situ hybridization. Mol Brain Res 53: 1–12, 1998.[Medline]
  31. Iwase K, Takemura M, Shimada T, and Wakisaka S. Ontogeny of NADPH-diaphorase in rat forebrain and midbrain. Anat Embryol (Berl) 197: 229–247, 1998.[CrossRef][Medline]
  32. Jaworski DM, Kelly GM, and Hockfield S. BEHAB, a new member of the proteoglycan tandem repeat family of hyaluronan-binding proteins that is restricted to the brain. J Cell Biol 125: 495–509, 1994.[Abstract/Free Full Text]
  33. Karanth S, Lyson K, and McCann SM. Role of nitric oxide in interleukin 2-induced corticotropin-releasing factor release from incubated hypothalami. Proc Natl Acad Sci USA 90: 3383–3387, 1993.[Abstract/Free Full Text]
  34. Kawakami S, Ichikawa M, Yokosuka M, Tsukamura H, and Maeda K. Glial and neuronal localization of neuronal nitric oxide synthase immunoreactivity in the median eminence of female rats. Brain Res 789: 322–326, 1998.[CrossRef][Web of Science][Medline]
  35. Kim SS, Kim H, Lee SJ, Chang HK, Shin MC, Jang MH, Shin MS, and Kim CJ. Treadmill exercise suppresses food-deprivation-induced increase of nitric oxide synthase expression in rat paraventricular nucleus. Neurosci Lett 353: 41–44, 2003.[CrossRef][Web of Science][Medline]
  36. Klatt P, Schmid M, Leopold E, Schmidt K, Werner ER, and Mayer B. The pteridine binding site of brain nitric oxide synthase. J Biol Chem 269: 13861–13866, 1994.[Abstract/Free Full Text]
  37. Knowles RG, Palacios M, Palmer RMJ, and Moncada S. Kinetic characteristics of nitric oxide synthase from rat brain. Biochem J 269: 207–210, 1990.[Web of Science][Medline]
  38. Konig JF and Klippel RA. The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Part of the Brainstem. Baltimore, MD: Williams & Williams, 1963.
  39. Lambert LE, Whitten JP, Baron BM, Cheng HC, Doherty NS, and McDonald IA. Nitric oxide synthesis in the CNS, endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues. Life Sci 48: 69–75, 1991.[CrossRef][Web of Science][Medline]
  40. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
  41. Mani SK, Allen JMC, Rettori V, McCann SM, O'Malley BW, and Clark JH. Nitric oxide mediates sexual behavior in female rats. Proc Natl Acad Sci USA 91: 6468–6472, 1994.[Abstract/Free Full Text]
  42. Marletta MA, Yoon PS, Iyengar R, Leaf CD, and Wishnock JS. Macrophage oxidation of L-arginine to nitrite and nitrate; nitric oxide is an intermediate. Biochemistry 27: 8706–8711, 1988.[CrossRef][Medline]
  43. Marshall HE, Hess DT, and Stamler JS. S-nitrosylation: physiological regulation of NF-{kappa}B. Proc Natl Acad Sci USA 101: 8841–8842, 2004.[Free Full Text]
  44. Marshall HE and Stamler JS. Inhibition of NF-{kappa}B by S-nitrosylation. Biochemistry 40: 1688–1693, 2001.[CrossRef][Medline]
  45. Mayer B, John M, Heinzel B, Werner ER, Wachter H, Schultz G, and Bohme E. Brain nitric oxide synthase is a biopterin- and flavin-containing multi-functional oxido-reductase. FEBS Lett 288: 187–191, 1991.[CrossRef][Web of Science][Medline]
  46. Modi S, Primrose WU, Lian LY, and Roberts GC. Effect of replacement of ferriprotoporphyrin IX in the haem domain of cytochrome P-450 BM-3 on substrate binding and catalytic activity. Biochem J 310: 939–943, 1995.[Medline]
  47. Moncada S, Radomski MW, and Palmer RMJ. Endothelium-derived relaxing factor; identification as nitric oxide and role in the control of vascular tone and platelet function. Biochem Pharmacol 37: 2495–2501, 1988.[CrossRef][Web of Science][Medline]
  48. Morley JE, Farr SA, Suarez MD, and Flood JF. Nitric oxide synthase inhibition and food intake: effects on motivation to eat and in female mice. Pharmacol Biochem Behav 50: 369–373, 1995.[CrossRef][Web of Science][Medline]
  49. Morley JE and Flood JF. Evidence that nitric oxide modulates food intake in mice. Life Sci 49: 707–711, 1991.[CrossRef][Web of Science][Medline]
  50. Morley JE and Flood JF. Competitive antagonism of nitric oxide synthetase causes weight loss in mice. Life Sci 51: 1285–1289, 1992.[CrossRef][Web of Science][Medline]
  51. Morley JE and Flood JF. Effect of competitive antagonism of NO synthastase on weight and food intake in obese and diabetic mice. Am J Physiol Regul Integr Comp Physiol 266: R164–R168, 1994.[Abstract/Free Full Text]
  52. Morley JE, Kumar VB, Mattammal M, and Villareal DT. Measurement of nitric oxide synthase and its mRNA in genetically obese (ob/ob) mice. Life Sci 57: 1327–1331, 1995.[CrossRef][Web of Science][Medline]
  53. Okamura H, Miyagawa A, Takagi H, Esumi H, Yanaihara N, and Ibata Y. Co-existence of PACAP and nitric oxide synthase in the rat hypothalamus. Neuroreport 5: 1177–1180, 1994.[Web of Science][Medline]
  54. Reid LS, Lim AR, and Mauk AG. Role of heme vinyl groups in cytochrome b5 electron transfer. J Am Chem Soc 108: 8197–8201, 1986.
  55. Rettori V, Belova N, Dees WL, Nyberg CL, Gimeno M, and McCann SM. Rose of nitric oxide in the control of luteinizing hormone-releasing hormone release in vivo and in vitro. Proc Natl Acad Sci USA 90: 10130–10134, 1993.[Abstract/Free Full Text]
  56. Rettori V, Gimeno M, Lyson K, and McCann SM. Nitric oxide mediates norepinephrine-induced prostaglandin E2 release from the hypothalamus. Proc Natl Acad Sci USA 89: 11543–11546, 1992.[Abstract/Free Full Text]
  57. Rettori V, Kamat A, and McCann SM. Nitric oxide mediates the stimulation of luteinizing-hormone releasing hormone release induced by glutamic acid in vitro. Brain Res Bull 33: 501–503, 1994.[CrossRef][Web of Science][Medline]
  58. Reynaert NL, Ckless K, Korn SH, Vos N, Guala AS, Wouters EFM, van der Vliet A, and Janssen YM. Nitric oxide represses inhibitory {kappa}B kinase through S-nitrosylation. Proc Natl Acad Sci USA 101: 8945–8950, 2004.[Abstract/Free Full Text]
  59. Rivier C and Shen GH. In the rat, endogenous nitric oxide modulates the response of the hypothalamic-pituitary-adrenal axis to interleukin-1beta, vasopressin, and oxytocin. J Neurosci 14: 1985–1993, 1994.[Abstract]
  60. Schulze A. Creatine deficiency syndromes. Mol Cell Biochem 244: 143–150, 2003.[CrossRef][Web of Science][Medline]
  61. Schuman EM and Madison DW. A requirement for the intracellular messenger nitric oxide in long term potentiation. Science 254: 1503–1506, 1991.[Abstract/Free Full Text]
  62. Sheta EA, McMillan K, and Masters BSS. Evidence for a bidomain structure of constitutive cerebellar nitric oxide synthase. J Biol Chem 269: 15147–15153, 1994.[Abstract/Free Full Text]
  63. Squadrito F, Calapai G, Altavilla D, Cucinotta D, Zingarelli B, Arcoraci V, Campo GM, and Caputi AP. Central serotoninergic system involvement in the anorexia induced by NG-nitro-L-arginine, an inhibitor of nitric oxide synthase. Eur J Pharmacol 255: 51–55, 1994.[CrossRef][Web of Science][Medline]
  64. Squadrito F, Calapai G, Altavilla D, Cucinotta D, Zingarelli B, Campo GM, Arcoraci V, Sautebin L, Mazzaglia G, and Caputi AP. Food deprivation increases brain nitric oxide synthase and depresses brain serotonin levels in rats. Neuropharmacology 33: 83–86, 1994.[CrossRef][Web of Science][Medline]
  65. Squadrito F, Calapai G, Altavilla D, Cucinotta D, Zingarelli B, Campo GM, Arcoraci V, Sautebin L, Mazzaglia G, and Caputi AP. Food deprivation increases brain nitric oxide synthase and depresses brain serotonin levels in rats. Neuropharmacology 33: 83–86, 1994.[CrossRef][Web of Science][Medline]
  66. Squadrito F, Calapai G, Cucinotta D, Altavilla D, Zingarelli B, Ioculano M, Urna G, Sardella A, Campo GM, and Caputi AP. Anorectic activity of NG-nitro-L-arginine, an inhibitor of brain nitric oxide synthase, in obese Zucker rats. Eur J Pharmacol 230: 125–128, 1993.[CrossRef][Web of Science][Medline]
  67. Summy-Long JY, Bui V, Mantx S, Koehler E, Weisz J, and Kadekaro M. Central inhibition of nitric oxide synthase preferentially augments release of oxytocin during dehydration. Neurosci Lett 152: 190–193, 1993.[CrossRef][Web of Science][Medline]
  68. Tamura M, Asakura T, and Yonetani T. Heme modification studies of myoglobin. I. Purification and some optical and EPR characteristics of synthesized myoglobins containing unnatural hemes. Biochim Biophys Acta 295: 467–479, 1973.[Medline]
  69. Turner MB, Corp ES, and Galbraith RA. Lack of NPY-induced feeding in cobalt protoporphyrin-treated rats is a postreceptor defect. Physiol Behav 56: 1009–1014, 1994.[CrossRef][Medline]
  70. Villar MJ, Ceccatelli S, Bedecs K, Bartfai T, Bredt D, Snyder SH, and Hokfelt T. Upregulation of nitric oxide synthase and galanin message-associated peptide in hypothalamic magnocellular neurons after hypophysectomy. Immunohistochemical and in situ hybridization studies. Brain Res 650: 219–228, 1994.[CrossRef][Web of Science][Medline]
  71. Yamada J, Sugimoto Y, Yoshikawa T, and Horisaka K. Effects of a nitric oxide synthase inhibitor on 5-HT1A receptor agonist 8-OH-DPAT-induced hyperphagia in rats. Eur J Pharmacol 316: 23–26, 1996.[CrossRef][Web of Science][Medline]
  72. Yamada J, Sugimoto Y, Yoshikawa T, and Horisaka K. Involvement of nitric oxide in 2-deoxy-D-glucose-induced hyperphagia in rats. Neuroreport 8: 2097–2100, 1997.[Web of Science][Medline]
  73. Yasin S, Costa A, Trainer P, Windle R, Forsling ML, and Grossman A. Nitric oxide modulates the release of vasopressin from rat hypothalamic explants. Endocrinology 133: 1466–1469, 1993.[Abstract/Free Full Text]
  74. Yonetani T, Yamamoto H, and Woodrow GVI. Studies on cobalt myoglobins and hemoglobins. J Biol Chem 249: 682–690, 1974.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
D. H. Kim, A. P. Burgess, M. Li, P. L. Tsenovoy, F. Addabbo, J. A. McClung, N. Puri, and N. G. Abraham
Heme Oxygenase-Mediated Increases in Adiponectin Decrease Fat Content and Inflammatory Cytokines Tumor Necrosis Factor-{alpha} and Interleukin-6 in Zucker Rats and Reduce Adipogenesis in Human Mesenchymal Stem Cells
J. Pharmacol. Exp. Ther., June 1, 2008; 325(3): 833 - 840.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
100/6/1983    most recent
01169.2005v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, M.
Right arrow Articles by Galbraith, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, M.
Right arrow Articles by Galbraith, R. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.