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CARDIOVASCULAR
-Melanocyte-Stimulating Hormone-Induced Hypotension in the Nucleus Tractus Solitarii of the Spontaneously Hypertensive Rats
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan (M.-H.T., W.-T.W., W.-C.L., J.Y.H.C., C.-J.L., H.-C.L., C.-J.T.); Graduate Institute of Biochemistry, Kaohsiung Medical University, Kaohsiung, Taiwan (M.-H.T.); and Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan (W.-T.W., C.-J.T.)
Received December 6, 2006; accepted February 2, 2007.
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Abstract |
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-melanocyte-stimulating hormone (-MSH), a POMC-derived neuropeptide, and its relationship with NO pathway in the NTS of spontaneously hypertensive rats (SHR). Unilateral microinjection of -MSH (0.3–300 pmol) into the NTS resulted in a dose-dependent hypotension and bradycardia in urethane-anesthetized SHR. The -MSH-induced hypotension was abolished by pretreatment with the antagonist of melanocortin-3/4 receptor (MC-3/4R), Ac-Nle-c[Asp-His-D-Nal(2')-Arg-Trp-Lys]-NH2 (SHU9119). Blockade of cAMP/protein kinase A (PKA), the downstream effector of melanocortin receptors, by previous injection of N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89) also ablated the cardiovascular effect of -MSH. To elucidate the role of NO pathway in -MSH-evoked hypotension, pretreatment with N
-nitro-L-arginine methyl ester, a universal inhibitor of nitric-oxide synthase (NOS), partially reversed the depressor and bradycardic effects of -MSH. Furthermore, previous application of the inducible NOS (iNOS) inhibitor, aminoguanidine, but not the neuronal NOS inhibitor, 7-nitroindazole, attenuated the cardiovascular effect of -MSH. Histological analysis revealed the colocalization of MC-4R, but not MC-3R, with iNOS in the NTS of SHR. In summary, intra-NTS injection of -MSH induces hypotension and bradycardia of SHR via MC-4R signaling, which activates cAMP/PKA and iNOS.
; Luger et al., 2003; Matsumura et al., 2003; Raffin-Sanson et al., 2003; Voisey et al., 2003; Catania et al., 2004; Ellacott and Cone, 2004; Cone, 2005). POMC is expressed at two locations in the brain: the arcuate nucleus of the hypothalamus and the nucleus tractus solitarii (NTS) of the brainstem (Ellacott and Cone, 2004; Cone, 2005). The NTS is a central site where various stimuli of baroreceptor reflexes and satiety integrate, thereby facilitating the cross-talk between cardiovascular regulation and energy homeostasis (Gordon, 1990; Appleyard et al., 2005; Cone, 2005). The POMC-derived neuropeptides in the NTS have been proposed to regulate the sympathetic activity and blood pressure (BP) (Li et al., 1996; Dunbar and Lu, 2000; Hill and Dunbar, 2002; Ramaekers et al., 2002; Kuo et al., 2003; Pavia et al., 2003).
-Melanocyte-stimulating hormone (-MSH) is an anorexic peptide derived from the POMC precursor and activates its receptors in brain, melanocortin-3 and -4 receptors (MC-3/4R), to cause hypophagia (Ellacott and Cone, 2004). Cumulative evidence shows that microinjection of -MSH or of its analogs into the central nervous system (CNS) regulates the food uptake and blood pressure (Li et al., 1996; Dunbar and Lu, 2000; Hill and Dunbar, 2002; Mizusawa et al., 2002; Pavia et al., 2003). However, there were conflicting views on cardiovascular function of -MSH in the CNS. Microinjection of -MSH into the medullary dorsal-vagal complex causes dose-dependent hypotension and bradycardia mediated by MC-4R (Li et al., 1996; Pavia et al., 2003). In contrast, intracerebroventricular administration of -MSH increases the lumbar sympathetic nerve activity and mean arterial pressure, which is reversed by pretreatment with the MC-4R antagonist, agouti protein (Dunbar and Lu, 2000; Hill and Dunbar, 2002; Mizusawa et al., 2002). The mechanism underlying the differential cardiovascular response of -MSH awaits to be delineated.
In this study, we investigated the cardiovascular effect of -MSH and activation of the central MC-3/4R in the NTS of spontaneously hypertensive rats (SHR), the most extensively used animal model for genetic hypertension and insulin resistance. Because nitric oxide (NO) plays an important role in cardiovascular regulation in the NTS (Tseng et al., 1996; Lo et al., 1998), the relationship of the -MSH-induced cardiovascular effect with NO pathway in the NTS of SHR was also investigated.
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Materials and Methods |
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-MSH and SHU9119 were purchased from Bachem Bioscience Inc. (King of Prussia, PA). Aminoguanidine (AG) and 7-nitroindazole (7-NI) were from Calbiochem (San Diego, CA). N-nitro-L-arginine methyl ester (L-NAME) and L-arginine were from Sigma Chemical (St. Louis, MO). All the drugs were dissolved in normal saline to the final concentrations in a volume not exceeding 60 nl. For each drug, only 60 nl was pressure-microinjected into the NTS.
Animals. The setup for intra-NTS microinjection has been described previously (Tseng et al., 1996; Lo et al., 1998). The animal studies were conducted in accordance to the Guidelines by the Committee of Animal Experiments, Kaohsiung Veterans General Hospital (Kaohsiung, Taiwan). The male SHR (weighing 280–340 g, 16–20 weeks old) were purchased from the Experimental Animal Center of National Science Council (Taipei, Taiwan). The rats were kept in individual cages in a room in which lighting was controlled (12 h on/12 h off), and temperature was maintained at 23–24°C. The rats were given Purina (St. Louis, MO) Laboratory Chow and tap water ad libitum.
The experimental procedures for intra-NTS microinjection has been described previously (Tseng et al., 1996; Lo et al., 1998). In brief, the rats were anesthetized with urethane (1.0 g/kg i.p. and 300 mg/kg i.v. if necessary) and placed in a stereotaxic frame; then the dorsal surface of the medulla was exposed. A glass micropipette cannula (0.031-inch o.d., 0.006-inch i.d.; Richland Glass Co., Vineland, NJ) was filled with test drugs or phosphate-buffered saline (PBS) and lowered into the NTS with A-P coordinates of 0.0 mm; M-L, 0.5 mm; and V, 0.4 mm with the obex used as reference. Injections were given over 10 s by air pressure generated by a hand-held syringe while the pipette tip was positioned in the NTS. The injection sites were confirmed by responsiveness to L-glutamate administration and histological confirmation. All the injections were unilateral.
The changes of BP and heart rate (HR) were measured intraarterially in anesthetized rats through unilateral microinjection of -MSH (0.3–300 pmol in 60 nl) into the NTS. The MC-3/4R antagonist (SHU9119, 250 pmol/60 nl), cAMP/protein kinase A (PKA) inhibitor (H89, 50 pmol/60 nl), NO donor (L-arginine, 50 nmol/60 nl), nonselective nitric-oxide synthase (NOS) inhibitor (L-NAME, 33 nmol/60 nl), inducible NOS (iNOS) inhibitor (AG, 2.5 nmol/60 nl), neuronal NOS (nNOS) inhibitor (7-NI, 2.5 nmol/60 nl), or vehicle was microinjected into the unilateral NTS alone or at 10 min before -MSH application.
Cell Cultures. Rat glioma C6 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 100 U/ml penicillin (Invitrogen), 100 µg/ml streptomycin (Invitrogen), 5% fetal calf serum (Invitrogen), and 2 mM glutamine (Invitrogen) under humidified conditions in 95% air and 5% CO2 at 37°C. Rat pituitary GH3 cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium (1:1) (Invitrogen) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 5% fetal calf serum, and 2 mM glutamine under humidified conditions in 95% air and 5% CO2 at 37°C.
Western Blot Analysis. C6 and GH3 cells lysates were prepared using buffer containing 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and protease inhibitors (Roche Applied Science, Indianapolis, IN). The rats were sacrificed with excessive dose of urethane, and the NTS tissues were dissected and immediately processed or stored at –70°C. The tissues were homogenized in lysis buffer and centrifuged at 12,000 rpm for 10 min, and the supernatant was collected to determine protein concentration with the Coomassie Blue plus protein assay reagent (Pierce, Rockford, IL). An aliquot of proteins (10 µg/ml) was separated by 10% SDS-polyacrylamide gel and transferred onto the polyvinylidene difluoride membranes (Immobilon-P membrane; Millipore, Bedford, MA). Membranes were blocked for 30 min with 5% skim milk in Tris-buffered saline/Tween 20 (10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% Tween 20) and incubated with antibodies against MC-4R (1: 1000; Chemicon Inc, Temecula, CA), MC-3R (1:1000; Santa Cruz Biotechnology Inc, Santa Cruz, CA), iNOS (1:500; BD Biosciences, La Jolla, CA), nNOS (1:1000; BD Biosciences), endothelial NOS (eNOS) (1:1000; BD Biosciences), and 
-actin (1:2000; Chemicon Inc). After washing with Tris-buffered saline/Tween 20, the membranes were then incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (1:5000; Vector Laboratories, Burlingame CA) for 60 min. Immunoreactivity was detected using the enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ).
Immunohistochemical Staining. The brainstem containing the NTS was fixed in fixation buffer (450 ml of 80% ethanol, 25 ml of acetic acid, and 50 ml of 37% formaldehyde), followed by embedding in paraffin and then cutting 5 µm thick per section. Immunohistochemical analysis for MC-4R, MC-3R, iNOS, nNOS, and eNOS detection was performed on the sections after paraffin was melted in an oven at 60°C for 1 h. The sections were deparaffinized in a xylene bath twice and rehydrated in decreasing grades of ethanol. Antigen retrieval was performed by heating the sections twice in a microwave for 5 min in 10 mM sodium citrate solution, pH 6.4. Following the microwave treatment, the sections were cooled to room temperature and then rinsed twice for 5 min in PBS. The endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide in methanol for 20 min and also rinsed twice for 5 min in PBS. Before incubation with the primary antibody, nonspecific binding was blocked by a reaction of the sections with 10% goat serum (Zymed Laboratories Inc, South San Francisco, CA) for 30 min and then rinsed twice for 5 min in PBS. Anti-MC-4R (1:200; Chemicon Inc), anti-MC-3R (1:200; Santa Cruz Biotechnology Inc), anti-nNOS (1: 500; BD Biosciences), and anti-eNOS (1:200; BD Biosciences) were incubated for 1 h at room temperature, whereas anti-iNOS (1:200; Chemicon Inc) was incubated at 4°C overnight. After incubation in the primary antibodies and rinsing twice for 5 min in PBS, the horseradish peroxidase/Fab polymer conjugate (Zymed Laboratories Inc) was added for 30 min to magnify the antibodies' signals. After rinsing three times with PBS, sections were stained with diaminobenzidine substrate (Vector Laboratories) for 5 min and counter-stained with H&E for 2 min to display the cell nucleus. Finally, the slides were mounted and observed under the light microscope.
Statistical Analysis. All the values are expressed as mean ± S.E.M. of indicated experiments. A paired t test (before and after pretreatments) was applied to compare group differences when significant main effects using a one-way analysis of variance were noted. The differences of p < 0.05 were considered significant.
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Results |
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-MSH Causes Dose-Dependent Depressor and Bradycardic Effects in SHR. To investigate the cardiovascular function of -MSH in hypertensive animals, -MSH (0.3–300 pmol) was unilaterally injected into the NTS of SHR. Intra-NTS injection of -MSH led to a reduction in mean BP (MBP) and HR in the urethane-anesthetized SHR at all of the dosages tested (Fig. 1A). Quantification analysis indicated that intra-NTS microinjection of -MSH induced a dose-dependent hypotension and bradycardia in SHR until doses higher than 30 pmol (Fig. 1B). Interestingly, microinjection of -MSH even at a dose as low as 0.3 pmol was sufficient to evoke significant hypotension of SHR (p < 0.05) (Fig. 1B). Because the maximal cardiovascular effect was achieved when 30 pmol of -MSH was administrated (–37 ± 5 mm Hg in MBP and –56 ± 5 beats/min in HR; n = 8), this concentration was used in the subsequent studies of SHR.
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Antagonist of MC-3/4R, SHU9119, Abolished the Cardiovascular Effect of -MSH in the NTS of SHR. Because MC-3R and MC-4R are the primary melanocortin receptors for -MSH in the CNS, we investigated whether blockade of MC-3/4R influenced the effect of -MSH in the NTS. It was found that previous injection of an MC-3/4R antagonist, SHU9119 (250 pmol), abolished the -MSH-induced decrease in BP and HR (from –36 ± 1 to –2 ± 3 mm Hg in MBP and –46 ± 4 to –1 ± 2 beats/min in HR, respectively; n = 8, p < 0.01) (Fig. 2). Moreover, the inhibition of -MSH-induced hypotension by SHU9119 was very potent and lasted for more than 90 min after drug delivery (Fig. 2A). These results suggested that blockade of MC-3/4R fully reverted the -MSH-induced hypotension and bradycardia in the NTS of SHR.
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-MSH in SHR. Because cAMP/PKA is the primary downstream effector in the signaling pathway of melanocortin receptors, we investigated whether PKA is involved in the -MSH-induced hypotensive effect using a specific PKA inhibitor, H89. Despite discernible effect in the NTS, injection of H89 (50 pmol) treatment potently ablated the cardiovascular depressive effect of -MSH (from –38 ± 8 to –5 ± 4 mm Hg in MBP and –56 ± 5 to –5 ± 3 beats/min in HR; n = 6, p < 0.01) (Fig. 3). Moreover, the inhibitory effect of H89 on -MSH-induced hypotension was sustained for more than 10 min (Fig. 3A). Therefore, the cAMP/PKA transmits the hypotensive signals for -MSH in the NTS of SHR.
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Pretreatment with a Universal NOS Inhibitor, L-NAME, Attenuated the Hypotensive and Bradycardic Effect of -MSH. To elucidate whether NO pathway affected the hypotensive and bradycardiac actions of -MSH, modulators of endogenous NO level such as a universal NOS inhibitor, L-NAME, or NO donor, L-arginine, were used. Pretreatment with L-NAME (33 nmol) significantly attenuated the depressor and bradycardic effects of -MSH (from –35 ± 8 to –17 ± 2 mm Hg in MBP and –51 ± 3 to –12 ± 6 beats/min in HR; n = 8, p < 0.05) (Fig. 4). Interestingly, application of L-arginine (50 nmol) prolonged the duration of -MSH-induced hypotension (from 2.0 ± 0.4 to 2.9 ± 0.5 min; n = 6, p < 0.05) but did not enhance the cardiovascular actions of -MSH in SHR (Supplementary Fig. 1). Together, these results suggest that the NO pathway participates in -MSH-induced hypotension in the NTS of SHR.
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-MSH-Mediated Cardiovascular Responses. There are three forms of NOS: eNOS, nNOS, and iNOS (Forstermann and Kleinert, 1995). To delineate the type(s) of NOS contributed to -MSH-evoked hypotension in the NTS, we investigated the effect of specific inhibitors of NOS on the -MSH-mediated hypotension. Intra-NTS injection of inhibitor of iNOS, AG (2.5 nmol), significantly attenuated the reduction in BP and HR (from –48 ± 9 to –20 ± 8 mm Hg in MBP and –45 ± 8 to –25 ± 4 beats/min in HR; n = 6, p < 0.05) (Fig. 5). In contrast, previous application of nNOS inhibitor, 7-NI (2.5 nmol), did not affect the -MSH-induced hypotension (Fig. 6). Thus, iNOS may be activated by intra-NTS -MSH administration to depress cardiovascular function of SHR.
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Colocalization of iNOS with MC-4R in the NTS of SHR. To further delineate the expression profile of NOS and MC-3/4R in the NTS of SHR, the tissue blocks and protein extracts were prepared from the NTS of SHR and analyzed for MC-3/4R and NOS expression by immunohistochemistry and Western blot analysis, respectively. Histological studies detected the expression of MC-4R, but not MC-3R, in the NTS of SHR (Fig. 7A). This observation was consistent with the findings of Western blot analysis, which revealed the presence MC-4R, but not MC-3R, in the NTS of SHR (Fig. 7B). To validate whether iNOS was indeed the downstream effector of MC-4R signaling, immunofluorescence analysis revealed that iNOS and MC-4R were colocalized in the NTS of SHR (Fig. 8A). Morphological analysis revealed that MC-4R and iNOS were present in the neurons of the NTS. This was consistent with the finding of Western blot analysis, which showed MC-4R was coexpressed with iNOS in the neuronal GH3 cells but not in glial C6 cells (Fig. 8B). Together, MC-4R may be the predominant receptor that transmits the hypotensive signals of -MSH in the NTS of SHR.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-MSH in the NTS of SHR. The present study shows for the first time that intra-NTS microinjection of -MSH potently induces hypotension in a genetically hypertensive SHR via the MC-4R pathway. Recently, Pavia et al. (2003) showed that administration of -MSH (250 pmol) or MTII (20–500 pmol), a potent agonist of MC-3/4R, into the NTS produced a rapid decrease in BP (approximately 10–12 mm Hg) and HR (approximately 18–23 beats/min) in Sprague-Dawley (SD) rats. Besides, they confirmed the role of MC-4R signaling in cardiovascular changes of -MSH using a selective antagonist of MC-4R, which is consistent with findings of the present study. Despite similar function and signaling pathway, there are some discrepancies in the -MSH studies between SHR and SD rats. First, the cardiovascular responses evoked by intra-NTS -MSH injection were greater in SHR. Second, a higher -MSH dose (approximately 100-1000-fold) was required to cause hypotension in SD rats compared with SHR. These data suggest that the genetically hypertensive SHR are more sensitive to intra-NTS -MSH application than the SD rats. Future studies are required to delineate whether the differential responses to -MSH also exist between SHR and the normotensive Wistar-Kyoto rats. In addition, it is imperative to investigate the expression of POMC/-MSH and melanocortin receptors in the NTS between SHR and Wistar-Kyoto rats.
Lack of MC-3R Expression in the NTS of SHR. One interesting finding is the identification of MC-4R, but not MC-3R, expression in the NTS of SHR. Both MC-3R and MC-4R expressed mainly in the CNS (Adan and Gispen, 1997; Gantz and Fong, 2003; Kuo et al., 2003; Voisey et al., 2003; Cone, 2005). The MC-3R has a wide distribution in the brain with high expression levels in medial areas of the hypothalamus, including the arcuate nucleus, whereas MC-4R is found only in and widely distributed in the cortex, thalamus, brain stem, spinal cord, and discrete nuclei in the medial hypothalamus. MC-4R receptor mRNA is abundantly expressed in the medullary dorsal vagal complex, an area that includes the dorsal motor nucleus of the vagus and the NTS (Li et al., 1996; Dunbar and Lu, 2000; Hill and Dunbar, 2002; Mizusawa et al., 2002; Pavia et al., 2003). The absence of MC-3R has been recently reported to cause 
-MSH resistance and hypertension in mice fed with high sodium diet (Ni et al., 2003). Because -MSH binds and transmits signals mainly through MC-3R, it will be also interesting to investigate the cardiovascular response to -MSH in SHR.
Signaling Circuits of -MSH in the NTS. The signaling pathway of -MSH in the NTS remains unclear. Because H89 effectively abrogated the -MSH-induced hypotension, the cAMP/PKA facilitated the predominant signaling effector after MC-4R activation by -MSH in the NTS of SHR. The involvement of NO pathway, particularly the iNOS signaling, in the -MSH-induced hypotension was strongly supported by the pharmaceutical intervention studies using NOS inhibitors such as L-NAME or AG. Because both AG and L-NAME attenuated the hypotensive function of -MSH by similar extent (50–60%), it seemed plausible that iNOS represented the main NOS activated by -MSH in the NTS of SHR. Although nNOS is relatively abundant in the NTS of SHR, the lack of influence on -MSH-induced hypotension by the nNOS inhibitor 7-NI suggested that nNOS was not likely involved (Chan et al., 2001, 2003). However, because application of L-NAME or AG did not completely revert the -MSH-mediated cardiovascular changes, alternative pathway(s) may exist downstream of cAMP/PKA to transmit the hypotensive signal of -MSH in the NTS.
Plausible Mechanism for iNOS Activation by -MSH in the NTS. It remains to be elucidated how -MSH activates iNOS in the NTS of SHR. During inflammatory responses, iNOS is activated by immunostimulating cytokines or bacterial pathogens to generate high concentrations of NO through the activation of nuclear factors such as nuclear factor 
B and activator protein-1 (Aktan, 2004). Recent evidence indicates that iNOS activation is regulated mainly at the transcriptional level but also at post-transcriptional, translational, and post-translational levels through effects on protein stability, dimerization, phosphorylation, cofactor binding, and availability of oxygen and L-arginine as substrates. Because of the lack of biochemical evidence, we could not conclude at which level the iNOS was activated by -MSH in the NTS of SHR. However, given that -MSH administration induces a rapid depressor effect in the NTS within less than 1 min, the mechanism of post-translational modification would be favored over the transcriptional regulation.
-MSH Differentially Regulates the NO Homeostasis. Central -MSH administration may differentially regulate the endogenous iNOS expression and activities, thereby altering the NO production in various cellular or animal models. Cumulative evidence indicates that -MSH treatment down-regulates the iNOS expression and activities (Star et al., 1995; Catania et al., 1999; Mandrika et al., 2001; Caruso et al., 2004). In peripheral tissues during endotoxin-induced sepsis, injection of -MSH inhibited the production and release of NO from macrophages (Star et al., 1995; Mandrika et al., 2001). Besides, the mRNA levels and the activities of iNOS in liver and lung were decreased by central injection of -MSH (Catania et al., 1999). During endotoxemia, -MSH reduces the induction of iNOS and cyclooxygenase-2 gene expression at the hypothalamic level and suggests that endogenous -MSH may exert an inhibitory tone on iNOS and cyclooxygenase-2 transcription via MC-4R acting as a local anti-inflammatory agent within the hypothalamus (Caruso et al., 2004). In contrast, there are reports supporting that -MSH treatment stimulates the NO production in vitro and in vivo. For example, intracerebroventricular injection of -MSH induces supraspinal erectile responses, which are mediated by NO and abolished by L-NAME administration (Mizusawa et al., 2002; Martin and MacIntyre, 2004). Likewise, despite the lack of effect on NO release, -MSH increases NO production in UV-irradiated melanocytes by induction of iNOS (Tsatmali et al., 2000). The differential functions of -MSH on NO homeostasis in different tissues and animal models remain to be elucidated. The present study indicates that -MSH microinjection into the NTS causes depressor and bradycardia effect in SHR via MC-4R and its downstream cAMP/PKA signaling pathway. Furthermore, -MSH may elicit NO production via iNOS pathway, which partially contributes to the hypotensive mechanism of -MSH in the NTS of SHR. In summary, the hypothesized signaling pathway for -MSH-evoked hypotension in the NTS of SHR is proposed (Supplementary Fig. 2). Future studies are required to dissect the molecular circuits between cAMP/PKA and iNOS in the NTS of SHR.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: POMC, pro-opiomelanocortin; NTS, nucleus tractus solitarii; BP, blood pressure; -MSH, -melanocyte-stimulating hormone; MC-3/4R, melanocortin-3/4 receptor(s); CNS, central nervous system; SHR, spontaneously hypertensive rat(s); NO, nitric oxide; SHU9119, Ac-Nle-c[Asp-His-D-Nal(2')-Arg-Trp-Lys]-NH2; AG, aminoguanidine; 7-NI, 7-nitroindazole; L-NAME, N-nitro-L-arginine methyl ester; PBS, phosphate-buffered saline; HR, heart rate; PKA, protein kinase A; H89, N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline; NOS, nitric-oxide synthase(s); iNOS, inducible nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; MBP, mean blood pressure; SD, Sprague-Dawley; MTII, cyclic -MSH analog; Ac-Nle4,Asp5,D-Phe7,Lys10-cyclo--MSH(4–10) amide.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Ching-Jiunn Tseng, Department of Medical Education and Research, Kaohsiung Veterans General Hospital, 386 Ta-Chung 1st Road, Kaohsiung 813, Taiwan. E-mail: cjtseng{at}vghks.gov.tw
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