Neuronal Nitric Oxide Synthase Activation Is Involved in Insulin-Mediated Cardiovascular Effects in the Nucleus Tractus Solitarii of Rats

doi:10.1016/j.neuroscience.2008.12.048

Neuroscience

Volume 159, Issue 2, 17 March 2009, Pages 727-734

https://doi.org/10.1016/j.neuroscience.2008.12.048 Get rights and content

Abstract

Neuronal nitric oxide synthases (nNOS) is distributed throughout the central nervous system (CNS) and has been proposed to modulate neuronal activity in the nucleus tractus solitarii (NTS). Here, we investigated whether the activation of nNOS is involved in insulin-induced cardiovascular responses in the NTS. Insulin (100 IU/ml) was unilaterally microinjected into the NTS, and the cardiovascular effects were evaluated before and after microinjection of the nNOS inhibitors 7-nitroindazole (7-NI) (5 pmol) and N(5)-(1-imino-3-butenyl)-l-ornithine (vinyl-L-NIO) (600 pmol). Western blot and immunohistochemical analyses were performed to determine nNOS phosphorylation levels after insulin or phosphoinositide 3-kinase (PI3K) inhibitor LY294002 microinjection into the NTS. Unilateral microinjection of insulin into the NTS produced prominent depressor and bradycardic effects in WKY rats. Pretreatment with the nNOS inhibitors 7-NI and Vinyl-L-NIO attenuated the cardiovascular response evoked by insulin in Wistar-Kyoto (WKY) rats. Moreover, Western blot analysis showed a significant increase in nNOS (16.5±0.4-fold; P<0.05; n=4) phosphorylation after insulin injection, whereas the PI3K inhibitor LY294002 abolished the insulin-induced effects. In situ nNOS phosphorylation was found to be increased in the NTS after insulin injection. Furthermore, co-immunoprecipitation assay showed Akt and nNOS can bind to each other as detected by phospho-Akt^S473 and phospho-nNOS^S1416 antibodies. In vitro kinase assay showed insulin activated Akt can directly phosphorylate nNOS^S1416. These results demonstrated that nNOS may couple with the activation of the insulin receptor, via the liberation of NO, in order to participate in central cardiovascular regulation of WKY rats.

Section snippets

Materials

Experimental drugs such as urethane, l-glutamate, and 7-NI were purchased from Sigma Chemical, Co. (St. Louis, MO, USA). Insulin was obtained from Novo Nordisk (Novo Nordisk A/S, Bagsværd, Denmark). Vinyl-L-NIO was purchased from Alexis (Alexis Corporation, Lausen, Switzerland). 7-NI, and vinyl-L-NIO were dissolved in Opti-MEM. All other drugs were dissolved in normal saline on the day of the experiment.

Animals and hemodynamic measurements

The Experimentation Ethics and Research Animal Facility Committee of Kaohsiung Veterans

Inhibitory effects of the nNOS inhibitors 7-NI and vinyl-L-NIO on cardiovascular regulation

In this study, we investigated whether the effect of nNOS in the NTS was responsible for the insulin-induced depressor and bradycardic responses. Unilateral microinjection (60 nl) of insulin (100 IU/ml) into the NTS produced prominent depressor and bradycardic effects in 8-week-old WKY rats (Fig. 1A). Pretreatment with 7-NI (5 pmol) significantly attenuated the cardiovascular effects of insulin in WKY rats (from −17±3 mm Hg and −42±10 bpm to −1±2 mm Hg and −1±1 bpm, respectively; P<0.05, paired

Discussion

In the present study, we demonstrated that microinjection of insulin into the NTS induced depressor and bradycardic effects. These results were similar to our previous findings (Huang et al., 2004). Our data suggest that insulin plays an important role in central cardiovascular regulation. Moreover, we showed that nNOS activation was required for insulin-mediated cardiovascular effects in the NTS of WKY rats. Both Western blot analysis and immunohistochemistry results showed a significant

Acknowledgments

This work was supported by grants awarded from the National Science Council (NSC94-2320-B-075B-002) and Kaohsiung Veterans General Hospital (VGHKS97-070) to Dr. C. J. Tseng.

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After blocking with bovine serum albumin solution (BSA) 5% in TBST buffer [TrisHCl: 20 mM pH 7,6; NaCl: 150 mM; Tween20: 0.01%], membranes were incubated with the primary antibody against NOS1 phosphorylated at Ser1417 residue (pNOS1; 1:1000, Abcam, UK, #ab5583), total nNOS (tNOS1; 1:500, Santa Cruz, USA, #sc5302) or GAPDH (1:5000, Santa Cruz, USA, #sc25778) for overnight at 4 °C. The phosphorylated Ser1417 residue is found in the active NOS1 following calcium influx [53–55]. Then, the membranes were incubated for 2 h at room temperature with secondary antibody against rabbit (Santa Cruz, #sc2313) or mouse (Santa Cruz, #sc2314) conjugated with peroxidase.
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NeuropharmacologyNeuronal Nitric Oxide Synthase Activation Is Involved in Insulin-Mediated Cardiovascular Effects in the Nucleus Tractus Solitarii of Rats

Abstract

Section snippets

Materials

Animals and hemodynamic measurements

Inhibitory effects of the nNOS inhibitors 7-NI and vinyl-L-NIO on cardiovascular regulation

Discussion

Acknowledgments

J Biol Chem

Regul Pept

Neurobiol Aging

J Biol Chem

J Chem Neuroanat

Life Sci

Neuroscience

Neuroscience

Prog Neurobiol

J Biol Chem

Am J Clin Nutr

Brain Res

Brain Res

Insulin and insulin-like growth factor receptors in the nervous system

Mol Neurobiol

Inhibition of rat cerebellar nitric oxide synthase by 7-nitro indazole and related substituted indazoles

Br J Pharmacol

Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase

Nature

Neuropharmacology
Neuronal Nitric Oxide Synthase Activation Is Involved in Insulin-Mediated Cardiovascular Effects in the Nucleus Tractus Solitarii of Rats