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

Neuroscience 159 (2009) 727–734

NEURONAL NITRIC OXIDE SYNTHASE ACTIVATION IS INVOLVED IN INSULIN-MEDIATED CARDIOVASCULAR EFFECTS IN THE NUCLEUS TRACTUS SOLITARII OF RATS H. T. CHIANG,a,b,c W. H. CHENG,a P. J. LU,d H. N. HUANG,a W. C. LO,a Y. C. TSENG,f J. L. WANG,e M. HSIAOe* AND C. J. TSENGa,b**

Key words: nitric oxide synthase, nucleus tractus solitarii, insulin, central cardiovascular regulation.

a Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan

The insulin receptor has been shown to be present in the CNS, which indicates the significant role of insulin in the CNS. It also has been demonstrated that insulin has an effect on cardiovascular function (Havrankova et al., 1978). Other reports showed the effects of insulin on arterial pressure and heart rate (HR) after peripheral injection and suggested that there is a function of insulin in central cardiovascular control (Werther et al., 1987, Edwards and Tipton, 1989). In the CNS, the nucleus tractus solitarius (NTS) is involved in regulating blood pressure (BP), HR, and sympathetic nerve activity (Dampney, 1994). The NTS receives inputs from afferent fibers arising from arterial baroreceptors, chemoreceptors, cardiopulmonary receptors, and other visceral receptors, and thus, has an important role in the autonomic control of the cardiovascular system (Dampney, 1994). In addition, insulin receptors are shown to be immunochemically positive in the NTS (Werther et al., 1987). We have reported that unilateral microinjection of insulin into the NTS produces predominant depressor and bradycardic effects. In situ AKT phosphorylation was found in the NTS by immunohistochemistry analysis after injection of insulin (Huang et al., 2004). These data suggested that insulin-mediated signaling in the NTS may play a significant role in the regulation of cardiovascular activity. Nitric oxide (NO) is synthesized enzymatically from l-arginine by nitric oxide synthase (NOS), which are either constitutive [epithelial (eNOS) and neuronal (nNOS)] or inducible (iNOS) isoforms (Knowles and Moncada, 1994). NO is a free radical gas that possesses multiple biological functions in the CNS and peripheral nervous system (Moncada et al., 1991). Previously, we have shown that microinjection of l-arginine into the NTS produced prominent depressor and bradycardic effects (Tseng et al., 1996; Lo et al., 1997, 1998, 2004). We also reported that inhibition of NOS attenuated baroreflex activation (Lo et al., 1996). These results suggested that NO formed by brain NOS in the NTS play significant roles in central cardiovascular regulation. nNOS has been demonstrated by immunohistochemical analysis to have high protein expression in the NTS (Zhang et al., 1998). It is an important component of transduction pathways that tonically inhibit the sympathetic outflow from the brain stem. It was reported recently that NO derived from nNOS contributes to the regulation of vaso-

b

Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan c

Chutung Veterans Hospital, Hsinchu County, Taiwan

d

Graduate Institute of Clinical Medicine, National Cheng-Kung University, Tainan, Taiwan e

Genomics Research Centre, Academia Sinica, Taipei, Taiwan

f

Department of Pharmacology, National Cheng Kung University, Tainan, Taiwan

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 (16.5ⴞ0.4-fold; P<0.05; nⴝ4) 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-AktS473 and phospho-nNOSS1416 antibodies. In vitro kinase assay showed insulin activated Akt can directly phosphorylate nNOSS1416. 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. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding authors. Tel: ⫹886-2-27899930-242; fax: ⫹886-227899931 (M. Hsiao), Tel: ⫹886-7-3422121-1505; fax: ⫹886-7-3468056 (C. J. Tseng). E-mail addresses: [email protected] (M. Hsiao), cjtseng@ vghks.gov.tw (C. J. Tseng). Abbreviations: BP, blood pressure; CNS, central nervous system; Co-IP, co-immunoprecipitation; eNOS, endothelial nitric oxide synthase; HR, heart rate; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; NTS, nucleus tractus solitarii; PI3K, phosphoinositide 3-kinase; Vinyl-L-NIO, N(5)-(1-imino-3-butenyl)-l-ornithine; WKY, Wistar-Kyoto; 7-NI, 7-nitroindazole. 0306-4522/09 © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.12.048

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motor tone and BP (Bredt et al., 1990). In addition, chronic administration of the specific nNOS inhibitor 7-nitroindazole (7-NI) in drinking water significantly increased BP in rats (Ollerstam et al., 1997). In contrast, bilateral microinjection of nNOS antisense oligonucleotides into the NTS can suppress nNOS immunoreactivity and increase BP (Maeda et al., 1999). Based on these findings, NO derived from nNOS may regulate cardiovascular effects in the NTS. Our recent study has demonstrated that the insulin– phosphoinositide 3-kinase (PI3K)–Akt–NOS signaling pathway was involved in cardiovascular regulation in the NTS (Huang et al., 2004). In order to clarify the function of NO induced by nNOS in regulating cardiovascular effects in the NTS, we investigated the effects of 7-NI, a relatively selective nNOS inhibitor (Babbedge et al., 1993), on cardiovascular response evoked by insulin in the NTS. Furthermore, another specific nNOS inhibitor N(5)-(1-imino-3butenyl)-l-ornithine (vinyl-L-NIO) (Babu and Griffith, 1998) was also used in this study. At the same time, we also demonstrated that in situ nNOS phosphorylation is induced by insulin in the NTS. Our findings indicated that nNOS is downstream factor in the insulin–PI3K–Akt signaling pathway, and that they mediate the central cardiovascular responses. They also suggested that phosphorylation of nNOS by the insulin–PI3K–Akt pathway, via the liberation of NO, participates in central cardiovascular regulation.

EXPERIMENTAL PROCEDURES 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 General Hospital had approved the animal protocols of this study. All efforts were made to minimize the number of animals used and their suffering. Animal studies were performed on male Wistar-Kyoto (WKY) rats that weighed 250 –300 g. Rats were anesthetized with urethane (1.0 g/kg i.p. and 300 mg/kg i.v. if necessary). The preparation of animals for intra-NTS microinjection and the methods used in localization of the NTS have been described previously (Tseng et al., 1996). A polyethylene cannula was inserted into the femoral vein for administration of drugs, and BP was measured directly via a cannula placed into the femoral artery and connected to a pressure transducer (P23 ID) and a polygraph (AT5000). HR was monitored continuously with a tachograph preamplifier (13-4615-65; Gould, Instrument Systems Inc., Valley View, OH, USA). A tracheostomy was performed to keep the airway patent during the experiment. For microinjections into the NTS, a glass cannula was filled with l-glutamate (0.154 nmol/60 nl) to functionally identify the NTS during the experiment. A specific decrease in BP and HR (ⱖ⫺35 mm Hg and ⫺50 bpm) was demonstrated after microinjection of l-glutamate in the NTS.

Central administration of insulin and drugs To investigate the effect of pre-administration of 7-NI (5 pmol/60 nl) on cardiovascular responses to insulin in the NTS, different groups of animals were injected with insulin (100 IU/ml, Novo Nordisk) into the unilateral NTS. The rats were then allowed to rest for at least 30 min until the mean BP and HR had returned to basal levels. After this, the changes in mean BP and HR were observed by microinjection of the same dose of insulin 10 min after intraNTS administration of 7-NI (50 pmol/60 nl) or vehicle. The cardiovascular action of the same dose of insulin was observed after 10 –90 min. Similar experimental procedures were used to study the effects of pretreatment with another specific nNOS inhibitor, vinylL-NIO (600 pmol/60 nl) on insulin in the NTS. The doses of the inhibitors (7-NI, LY294002, vinyl-L-NIO) were chosen with neither toxic nor inducing prominent pressor or depressor effects as described previously (Lin et al., 1999; Huang et al., 2004; Chiang et al., unpublished observations).

Western blot analysis The NTS tissues were separated carefully under the microscopic examination from individual rats. Briefly, tissues on both sides of the dorsomedial part of the medulla oblongata at the level of NTS (1 mm rostral or caudal from the obex) were collected by micropunches made with a stainless steel bore (1 mm i.d.) (Huang et al., 2004). Individual NTS tissues with or without insulin and LY294002 injections were immediately excised 5–10 min after the injection for protein extraction. Total protein was prepared by homogenizing the NTS in lysis buffer containing 20 mmol/l imidazole–HCl (pH 6.8), 100 mmol/l KCl, 2 mmol/l MgCl2, 20 mmol/l EGTA (pH 7.0), 300 mmol/l sucrose, 1 mmol/l NaF, 1 mmol/l sodium vanadate, 1 mmol/l sodium molybdate, 0.2% Triton X-100, and a proteinase inhibitor cocktail (Roche, Basel, Switzerland) for 1 h at 4 °C. Proteins extracted (30 ␮g per sample assessed by BCA protein assay) (Pierce Chemical, Co., Rockford, IL, USA) were subjected to 7.5%–10% SDS–Tris glycine gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare, Buckingamshire, UK). The membrane was blocked with 5% nonfat milk in PBST buffer (10 mmol/l Tris [pH 7.5], 100 mmol/l NaCl, 0.1% Tween 20), incubated with anti-phospho-ser1416 nNOS antibody (Abcam, Cambridge, UK), at 1:2000, in PBST with bovine serum albumin, and incubated for 1 h at room temperature. Peroxidase-conjugated antimouse or anti-rabbit antibody (1:5000) was used as a secondary antibody. The membrane was developed using the ECL-Plus protein detection kit (GE Healthcare).

Immunohistochemistry analysis Immunohistochemical staining was performed to determine whether nNOS phosphorylation occurred in situ after insulin injections in the NTS of WKY rats. The rat brain was collected 5–10 min after the insulin injection and was fixed with 4% formaldehyde. Paraffin-embedded serial sections were cut at 5 ␮m thickness. The sections were dewaxed, quenched in H2O2/methanol, microwaved in citric buffer (pH 6.0), blocked in 5% goat serum, and incubated in anti-p-nNOSS1416 (Abcam) antibody (1:50) at 4 °C overnight. Afterward, sections were incubated in biotinylated secondary antibody (1:200) for 1 h and in AB complex (1:100) for 30 min in room temperature. The sections were visualized using the DAB substrate kit (Vector Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin. The sections were then photographed with a microscope equipped with a CCD camera.

Co-immunoprecipitation (Co-IP) assay The NTS was dissected by micropunch (1-mm inner diameter) from a 1-mm thick brainstem slice at the level of obex under a

H. T. Chiang et al. / Neuroscience 159 (2009) 727–734 microscope (Huang et al., 2004). Total protein were prepared by homogenizing the NTS tissue in lysis buffer containing 20 mM imidazole–HCl (pH 6.8), 100 mM KCl, 2 mM MgCl, 20 mM EGTA (pH 7.0), 300 mM sucrose, 1 mM NaF, 1 mM sodium vanadate, 1 mM Na molybdate, 0.2% Triton X-100, and proteinase inhibitor cocktail (Roche) for 1 h at 4 °C. The resulting supernatant was incubated with 5 ␮l rabbit anti-nNOS (Upstate Biotechnology, Lake Placid, NY, USA), anti-Akt (Cell Signaling Technology, Danvers, MA, USA) antibodies, and subjected to immunoprecipitation by using Catch and Release v2.0 kit (Upstate Biotechnology), according to the manufacturer’s protocol. The proteins binding to capture resins were eluted by boiling in 2⫻ SDS-sample buffer, separated by 7.5% SDS-PAGE, and subjected to Western blot analysis using anti-p-nNOSS1416 (Abcam), and anti-p-AktS473 (Cell Signaling Technology).

In vitro kinase assay For the in vitro kinase assay, a Co-IP experiment using anti-Akt (Cell Signaling Technology) and anti-nNOS (Upstate Biotechnology) antibodies were performed and the Akt-nNOS complex was eluted by Catch and Release v2.0 kit (Upstate Biotechnology). The kinase reaction was initiated by the addition of kinase buffer (100 mM Hepes, pH 7.4, 1 mM DTT, 10% glycerol, 100 mM ATP, 1 M MgCl2), and the phosphorylation reactions were terminated with the addition of 2⫻ SDS-sample buffer and boiled for 10 min. The phosphorylation of nNOS was determined by Western blot analysis using anti-p-nNOSS1416 (Abcam), anti-nNOS (Upstate Biotechnology) antibodies.

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7-NI, and vinyl-L-NIO) as a sham control in place of 7-NI, or vinyl-L-NIO did not modify the cardiovascular effects of insulin (data not shown). In situ nNOS phosphorylation induced by insulin injection in the NTS We then investigated whether nNOS was activated after insulin injection into NTS. Phosphorylation level of nNOS was determined by Western blot analysis using antibodies specific for phospho-nNOSS1416. The results in Fig. 3A show a significant increase in eNOS phosphorylation after insulin injection into the NTS as compared to control (Fig. 3A, lane 2 vs. lane 1; 16.5⫾0.4-fold; P⬍0.05). More importantly, the significant increase in nNOS phosphorylation induced by insulin can be blocked by LY294002, the specific inhibitor of PI3K (Vlahos et al., 1994) (Fig. 3A, lane 3, and Fig. 3B). We further determined whether nNOS phosphorylation occurred in situ after insulin injection. Paraffin sections of the NTS were subjected to immunohistochemical staining analysis with phospho-nNOS antibodies. Fig. 3C showed strong in situ nNOS phosphorylation in the NTS after insulin microinjection. Insulin activated Akt binds and phosphorylates nNOS at Ser1416

Statistical analysis A paired t-test (before and after pretreatments) or Dunnett’s test was applied to compare group differences when significant effects were noted by one-way ANOVA. Differences with a probability value ⬍0.05 were considered significant. All data are expressed as mean⫾SEM.

RESULTS 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 t-test; n⫽16; Fig. 1A, B). The attenuation effect of 7-NI lasted for more than 90 min. This finding was further supported using vinyl-L-NIO. Fig. 2A shows that prior administration of vinyl-L-NIO (600 pmol) significantly attenuated the cardiovascular responses evoked by insulin in 8-week-old WKY rats (from ⫺22⫾6 mm Hg and ⫺48⫾14 bpm to ⫺5⫾2 mm Hg and ⫺5⫾2 bpm, respectively; P⬍0.05, paired t-test; n⫽8; Fig. 2B). The attenuated effects caused by vinyl-L-NIO on insulin-induced cardiovascular response reached a plateau status 10 min after insulin injection and returned to depressor and bradycardic effects at 30 or 60 min (Fig. 2A). In comparison, administration of Opti-MEM (the solvent of

The phosphorylation of Ser1416 of nNOS was reported as one of the in vivo phosphorylation sites that is required for its activation (Fulton et al., 1999; Garcin et al., 2004). The kinase responsible for Ser1416 phosphorylation however remains unclear. To determine which kinase is responsible for phosphorylating nNOSS1416, we used two bioinformatic Web sites: the NetPhosK 1.0 Server (http://www.cbs. dtu.dk/services/NetPhosK) and kinase Phos 2.0 (http:// kinasephos2.mbc.nctu.edu.tw) to predict interrelated proteins. The results suggested that Akt could phosphorylate nNOSS1416, having scores of 0.53 and 0.51. We then performed Co-IP assays to analyze whether Akt binds to nNOS. Our results show that nNOS immunoprecipitated by specific nNOS antibodies can precipitate P-Akt 473 from NTS lysate; immunoprecipitated Akt was also observed to interact with P-nNOS 1416 (Fig. 4A). In vitro kinase assay was then performed to determine whether Akt can directly phosphorylate nNOSS1416 in vitro. We incubated the purified nNOS with the Akt immunoprecipitated from NTS lysate in the presence of ATP for the reaction time as indicated. Fig. 4B and 4C shows that phosphorylation at Ser1416 of nNOS by Akt increases in a time dependent manner (from 0 min to 120 min). Thus, these results demonstrate that Akt can directly phosphorylate nNOS at Ser1416 in vitro and maybe in the NTS.

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. More-

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Fig. 1. Cardiovascular effects of unilateral injection of insulin (100 IU/ml) into the NTS before and after 7-NI (5 pmol) injection in 8-week-old WKY rats. (A) Insulin and 7-NI were injected at the indicated time points. MBP represents mean BP. HR was recorded at a paper speed of 3 mm/min. The horizontal bar represents recording over 5-min intervals. (B) Comparative MBP and HR effects of insulin (100 IU/ml) and the nNOS inhibitor 7-NI (5 pmol) on unilateral intra-NTS administration of substances in WKY rats. Insulin was injected in the absence (control) or presence of 7-NI. The vertical bars represent SEM change from baseline values. Each bar represents the average data from eight rats. * P⬍0.05, compared with the control value.

over, 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 increase in nNOSS1416 phosphorylation after insulin injection. These results suggest that the activation of nNOS is involved in insulinmediated cardiovascular responses in the NTS.

Insulin receptors have been known to be widely expressed in the CNS (Adamo et al., 1989; Unger et al., 1991), and they are unevenly distributed throughout the brain, with a particularly high density in the cerebral cortex, olfactory bulb, hippocampus, cerebellum, and hypothalamus. Insulin receptor immunoreactivity is found in both neurons and glial cells (Unger et al., 1991; Baskin et al.,

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Fig. 2. Cardiovascular effects of unilateral injection of insulin (100 IU/ml) into the NTS before and after vinyl-L-NIO (600 pmol) injection into 8-week-old WKY rats. (A) Insulin and vinyl-L-NIO were injected at indicated time points. MBP represents mean BP. HR was recorded at a paper speed of 3 mm/min. The horizontal bar represents recording over 5-min intervals. (B) Comparative MBP and HR effects of insulin (100 IU/ml) with the nNOS-specific inhibitor vinyl-L-NIO (600 pmol) on unilateral intra-NTS administration of substances in WKY rats. Insulin was injected in the absence (control) or presence of vinyl-L-NIO. The vertical bars represent SEM change from baseline values. Each bar represents the average data from eight rats. * P⬍0.05, compared with the control value.

1993). Insulin is transported into the brain from peripheral tissues via the cerebrospinal fluid (CSF) (Woods et al., 1985) and may be synthesized by neurons in the brain (Craft et al., 1996). Although insulin receptors are widely dispersed throughout tissues of the periphery, and their function is well known, the existence and the function of insulin receptors within the brain are somewhat of an enigma. Evidence included this study, however, has sug-

gested that insulin/NO signaling may exist in the NTS and plays a significant role in regulating cardiovascular activity. The NTS is the primary integrative center for cardiovascular control and other autonomic functions in the CNS. Our previous studies have demonstrated that NO is involved in central cardiovascular regulation, and that it modulates sympathetic nervous activity and the baroreflex in the NTS (Tseng et al., 1996; Lo et al., 1997, 1998, 2004). We

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Fig. 3. Western blot and immunohistochemical analysis of nNOS phosphorylation in the NTS after insulin injection. (A) Western blot depicts abundant p-nNOS protein in the NTS of WKY rats after insulin microinjection (lane 2). LY294002 blocks insulin-induced expression of p-nNOS in WKY rats (lane 3). (B) Densitometric analysis of p-nNOS and nNOS levels before and after insulin and LY294002 injection. Bars are mean⫾SE of four independent experiments. * P⬍0.05 vs. lane 1 and # P⬍0.05 vs. lane 2. (C) In situ nNOS phosphorylation after insulin injection into the NTS of WKY rats was demonstrated by immunohistochemical staining. (C.b) p-nNOS immunostaining in the NTS with p-nNOSS1416 antibody. Arrowheads indicate cells with nNOS phosphorylation after insulin injection compared with control (C.a). Scale bar⫽50 ␮m.

reported recently that the insulin-signaling pathway might couple to activation of NOS to participate in central cardiovascular regulation, and there may be an interaction between insulin and PI3K–Akt–NOS–NO-mediated signaling in the NTS of WKY rats (Huang et al., 2004). However, it was not clear whether nNOS was mediated by insulin. To clarify this, we used inhibitors of nNOS. Pretreatment with the nNOS inhibitors 7-NI and vinyl-L-NIO attenuated the cardiovascular responses evoked by insulin in WKY rats (Figs. 1 and 2). In addition, Western blot and immunohistochemical analyses were performed to determine eNOS and nNOS phosphorylation levels after insulin or PI3K inhibitor (LY294002) microinjection into the NTS. Western blot analysis showed a significant increase in nNOSS1416 phosphorylation after insulin injection, whereas LY294002 abolished the insulin-induced effects (Fig. 3A, B). In situ nNOS phosphorylation was increased after injection of insulin by immunohistochemical analysis in the NTS (Fig. 3C). Taken together, our data suggested that the activation of nNOS by insulin might be mediated through NO in the NTS of WKY rats. It has been indicated that, under physiological conditions, insulin may regulate the expression of nNOS in the brain (Yuan et al., 2004). In support of pharmacological

and physiological studies for a role of nNOS in cardiovascular signal transmission in the NTS, nNOS has been found in neurons and fibers of the NTS and in vagal afferents and their terminals in the NTS (Lawrence et al., 1998; Lin et al., 1998, 2000). It has also been demonstrated that exogenous insulin could upregulate the expression and activity of nNOS in R2 cells, cerebral cortical astrocytes, and neurons of rats (Yuan et al., 2004). It is well known that nNOS can be phosphorylated by many kinases including cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC), and calcium-calmodulin protein kinase II (CaMK II) (Bredt et al., 1991). In fact, it has been shown that nNOS Ser1416 is an Akt-dependent phosphorylation motif in its reductase domain that might regulate its activation (Fulton et al., 1999). In the present study, prior administration of nNOS inhibitor 7-NI attenuated the cardiovascular effects induced by insulin (Fig. 1). And, pretreatment with another specific nNOS inhibitor vinyl-L-NIO also attenuated the cardiovascular responses of insulin (Fig. 2). The phosphorylation of nNOS at Ser1416 was highly elevated after insulin microinjection into the NTS (Fig. 3); we also demonstrated nNOS Ser1416 phosphorylation in situ in the NTS by immunohistochemical staining. LY294002 inhibited nNOS Ser1416

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and Kaohsiung Veterans General Hospital (VGHKS97-070) to Dr. C. J. Tseng.

REFERENCES

Fig. 4. Insulin stimulates Akt kinase activity and nNOS function. Akt is a PI3K-regulated kinase that can phosphorylate nNOS at Ser1416 in the NTS. (A) Lysates from NTS were assayed and used Co-IP of Akt and nNOS. Total lysates were also analyzed by immunoblot with antibodies of phosphorylated Akt and nNOS in the experiment. (B) Akt-associated kinase assay was determined using nNOS as a substrate. Phosphorylated nNOS were analyzed by 7.5% SDS–PAGE and autoradiography. (C) The histogram shows the phosphorylation ratio of nNOS compared with the time of incubation in Akt and ATP.

phosphorylation in Western blot analysis. Our data showed that after administration of insulin, we could find the interaction between Akt and nNOS by Co-IP assay. Furthermore, Akt could directly increase nNOS phosphorylation at Ser1416 in a time-dependent procedure in vitro. In this study, we confirmed and extended our previous observation that nNOS participate in the effect of insulin on cardiovascular modulation in the NTS. Lin et al. (1998) have found eNOS and nNOS in cells and processes in all NTS subnuclei, and they suggest that NO produced by nNOS may directly modulate neurotransmission in the NTS. In conclusion, these results demonstrated that nNOS may be a downstream factor of the insulin–PI3K–Akt signaling pathway–mediated cardiovascular response in the NTS of WKY rats. They also suggested that phosphorylation of nNOS by the insulin–PI3K–Akt pathway, via the liberation of NO, participates in central cardiovascular regulation. Acknowledgments—This work was supported by grants awarded from the National Science Council (NSC94-2320-B-075B-002)

Adamo M, Raizada MK, LeRoith D (1989) Insulin and insulin-like growth factor receptors in the nervous system. Mol Neurobiol 3: 71–100. Babbedge RC, Bland-Ward PA, Hart SL, Moore PK (1993) Inhibition of rat cerebellar nitric oxide synthase by 7-nitro indazole and related substituted indazoles. Br J Pharmacol 110:225–228. Babu BR, Griffith OW (1998) N5-(1-imino-3-butenyl)-L-ornithine. A neuronal isoform selective mechanism-based inactivator of nitric oxide synthase. J Biol Chem 273:8882– 8889. Baskin DG, Sipols AJ, Schwartz MW, White MF (1993) Immunocytochemical detection of insulin receptor substrate-1 (IRS-1) in rat brain: colocalization with phosphotyrosine. Regul Pept 48:257– 266. Bredt DS, Hwang PM, Glatt CE, Lowenstein C, Reed RR, Snyder SH (1991) Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 351:714 –718. Bredt DS, Hwang PM, Snyder SH (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347: 768 –770. Craft S, Newcomer J, Kanne S, Dagogo-Jack S, Cryer P, Sheline Y, Luby J, Dagogo-Jack A, Alderson A (1996) Memory improvement following induced hyperinsulinemia in Alzheimer’s disease. Neurobiol Aging 17:123–130. Dampney RA (1994) Functional organization of central pathways regulating the cardiovascular system. Physiol Rev 74:323–364. Edwards JG, Tipton CM (1989) Influences of exogenous insulin on arterial blood pressure measurements of the rat. J Appl Physiol 67:2335–2342. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC (1999) Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399:597– 601. Garcin ED, Bruns CM, Lloyd SJ, Hosfield DJ, Tiso M, Gachhui R, Stuehr DJ, Tainer JA, Getzoff ED (2004) Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase. J Biol Chem 279:37918 –37927. Havrankova J, Roth J, Brownstein M (1978) Insulin receptors are widely distributed in the central nervous system of the rat. Nature 272:827– 829. Huang HN, Lu PJ, Lo WC, Lin CH, Hsiao M, Tseng CJ (2004) In situ Akt phosphorylation in the nucleus tractus solitarii is involved in central control of blood pressure and heart rate. Circulation 110: 2476 –2483. Knowles RG, Moncada S (1994) Nitric oxide synthases in mammals. Biochem J 298 (Pt 2):249 –258. Lawrence AJ, Castillo-Melendez M, McLean KJ, Jarrott B (1998) The distribution of nitric oxide synthase-, adenosine deaminase- and neuropeptide Y-immunoreactivity through the entire rat nucleus tractus solitarius: effect of unilateral nodose ganglionectomy. J Chem Neuroanat 15:27– 40. Lin HC, Wan FJ, Cheng KK, Tseng CJ (1999) Nitric oxide signaling pathway mediates the L-arginine-induced cardiovascular effects in the nucleus tractus solitarii of rats. Life Sci 65:2439 –2451. Lin LH, Cassell MD, Sandra A, Talman WT (1998) Direct evidence for nitric oxide synthase in vagal afferents to the nucleus tractus solitarii. Neuroscience 84:549 –558. Lin LH, Emson PC, Talman WT (2000) Apposition of neuronal elements containing nitric oxide synthase and glutamate in the nucleus tractus solitarii of rat: a confocal microscopic analysis. Neuroscience 96:341–350. Lo WC, Hsiao M, Tung CS, Tseng CJ (2004) The cardiovascular effects of nitric oxide and carbon monoxide in the nucleus tractus solitarii of rats. J Hypertens 22:1182–1190.

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Lo WC, Jan CR, Wu SN, Tseng CJ (1998) Cardiovascular effects of nitric oxide and adenosine in the nucleus tractus solitarii of rats. Hypertension 32:1034 –1038. Lo WC, Lin HC, Ger LP, Tung CS, Tseng CJ (1997) Cardiovascular effects of nitric oxide and N-methyl-D-aspartate receptors in the nucleus tractus solitarii of rats. Hypertension 30:1499 –1503. Lo WJ, Liu HW, Lin HC, Ger LP, Tung CS, Tseng CJ (1996) Modulatory effects of nitric oxide on baroreflex activation in the brainstem nuclei of rats. Chin J Physiol 39:57– 62. Maeda M, Hirano H, Kudo H, Doi Y, Higashi K, Fujimoto S (1999) Injection of antisense oligos to nNOS into nucleus tractus solitarii increases blood pressure. Neuroreport 10:1957–1960. Moncada S, Palmer RM, Higgs EA (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43:109 –142. Ollerstam A, Pittner J, Persson AE, Thorup C (1997) Increased blood pressure in rats after long-term inhibition of the neuronal isoform of nitric oxide synthase. J Clin Invest 99:2212–2218. Tseng CJ, Liu HY, Lin HC, Ger LP, Tung CS, Yen MH (1996) Cardiovascular effects of nitric oxide in the brain stem nuclei of rats. Hypertension 27:36 – 42.

Unger JW, Livingston JN, Moss AM (1991) Insulin receptors in the central nervous system: localization, signalling mechanisms and functional aspects. Prog Neurobiol 36:343–362. Vlahos CJ, Matter WF, Hui KY, Brown RF (1994) A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1benzopyran-4-one (LY294002). J Biol Chem 269:5241–5248. Werther GA, Hogg A, Oldfield BJ, McKinley MJ, Figdor R, Allen AM, Mendelsohn FA (1987) Localization and characterization of insulin receptors in rat brain and pituitary gland using in vitro autoradiography and computerized densitometry. Endocrinology 121:1562– 1570. Woods SC, Porte D Jr, Bobbioni E, Ionescu E, Sauter JF, RohnerJeanrenaud F, Jeanrenaud B (1985) Insulin: its relationship to the central nervous system and to the control of food intake and body weight. Am J Clin Nutr 42:1063–1071. Yuan ZR, Liu B, Zhang Y, Yuan L, Muteliefu G, Lu J (2004) Upregulated expression of neuronal nitric oxide synthase by insulin in both neurons and astrocytes. Brain Res 1008:1–10. Zhang K, Zucker IH, Patel KP (1998) Altered number of diaphorase (NOS) positive neurons in the hypothalamus of rats with heart failure. Brain Res 786:219 –225.

(Accepted 27 December 2008) (Available online 3 January 2009)