Neuronal nitric oxide synthase activation is involved in insulin-mediated cardiovascular effects in the nucleus tractus solitarii of rats.
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.
Chiang
H T
HT
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
Cheng
W H
WH
Lu
P J
PJ
Huang
H N
HN
Lo
W C
WC
Tseng
Y C
YC
Wang
J L
JL
Hsiao
M
M
Tseng
C J
CJ
D000704:Analysis of Variance; D000818:Animals; D001794:Blood Pressure / Q000187:drug effects*; D002867:Chromones / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006339:Heart Rate / Q000187:drug effects* / Q000528:radiation effects; D047468:Immunoprecipitation / Q000379:methods; D007191:Indazoles / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D008845:Microinjections / Q000379:methods; D009025:Morpholines / Q000494:pharmacology; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D009952:Ornithine / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D011921:Rats, Inbred WKY; D017552:Solitary Nucleus / Q000187:drug effects* / Q000201:enzymology
D002867:Chromones; D004791:Enzyme Inhibitors; D007191:Indazoles; D007328:Insulin; D009025:Morpholines; C111862:N(5)-(1-imino-3-butenyl)ornithine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D009952:Ornithine; D052248:Nitric Oxide Synthase Type I; C080122:7-nitroindazole