臺灣博碩士論文加值系統

摘要


磷脂質PI(3,4,5)P3在PI3K細胞訊息傳遞路徑中是扮演第二傳訊者(second messenger)的角色。胰島素(insulin)刺激血管內皮細胞產生一氧化氮(nitric oxide)進而使血管舒張及增加血流量的調控機制也涉及PI3K細胞訊息傳遞路徑。然而一氧化氮也曾經被報導過產生在神經細胞裡面，在此一氧化氮的作用是神經傳導物質(neurotransmitter)。此外，一氧化氮也被報導在孤立束核(NTS)的中樞血壓調控裡扮演著重要的角色。先前在我們實驗室裡，將胰島素微注射到大鼠的孤立束核裡會造成明顯的血壓降低以及心博舒緩的現象，另外也會造成PI3K下游的蛋白激酶Akt的活化。為了研究胰島素在孤立束核裡造成一氧化氮產生更詳細的下游訊息傳遞機制，我們在神經細胞株PC12和GH3裡以及在大鼠的孤立束核裡，研究脂類第二傳信者- PI(3,4,5)P3對一氧化氮產生的影響。將PC12和GH3細胞以10 �嵱 PI(3,4,5)P3處理後，Akt的磷酸化和一氧化氮的產生都增加了。將1 mM PI(3,4,5)P3微注射到SD大鼠的孤立束核裡也會造成血壓降低以及心博舒緩的現象，而且也造成Akt磷酸化的增加。此外，為了研究高血壓的分子致病機轉，我們比較在自發性高血壓大鼠(SHR)和正常血壓的控制組WKY大鼠對PI(3,4,5)P3造成的心血管反應的不同。研究結果顯示，和SHR比較起來，PI(3,4,5)P3在WKY大鼠裡會造成時間較久的血壓下降反應以及在16周大的WKY大鼠會有較大的心博舒緩的反應。 綜合以上結果，PI(3,4,5)P3活化Akt訊息在孤立束核對中樞心血管調控裡扮演一個重要的角色。此外，在自發性高血壓大鼠裡可能是PI(3,4,5)P3的下游基因有缺陷以致於產生高血壓。

Neurogranin (Ng)和neuromodulin (Nm)是一種只在神經細胞表現的蛋白質。Ng和Nm平常是和calmodulin鍵結在一起，因此Ng和Nm可能參與了鈣離子造成的一氧化氮產生的調控。此外，由於Ng和Nm對PI(3,4,5)P3的高親合力，所以Ng和Nm也可能調控PI(3,4,5)P3引發一氧化氮產生之作用。在此研究裡，我們純化了Ng和Nm的合成蛋白及其多株抗體。將Ng (0.3 �慊/�愮)或Nm (0.3 �慊/�愮)微注射到SD大鼠的孤立束核並沒有造成任何的心血管反應。然而，若先將Ng或Nm微注射到SD大鼠的孤立束核則會減弱PI(3,4,5)P3的心血管反應。因此，Ng和Nm在孤立束核裡可能會調控PI(3,4,5)P3的心血管反應。

Abstract


The phospholipid PI(3,4,5)P3 works as a second messenger in PI3K signaling pathway. The PI3K signaling pathway is involved in insulin stimulated nitric oxide (NO) production in vascular endothelium, leading to vasodilation and increased blood flow. However, the production of NO also has been reported in neurons as a neurotransmitter and in nucleus tractus solitarii (NTS), NO plays a role in central cardiovascular regulation. Previously, microinjection of insulin into the NTS of rats produces prominent depressor and bradycardic and activates the PI3K downstream Akt. Therefore, to investigate the detail downstream signaling of insulin stimulated NO production in NTS, the effects of PI(3,4,5)P3 on NO production were determined in neuronal cell lines PC12 and GH3 and in NTS of SD rats. The GH3 and differentiated PC12 exposed to 10�嵱 PI(3,4,5)P3 showed an increase in Akt phosphorylation and in NO production. Microinjection of 1 mM PI(3,4,5)P3 into the NTS of SD rats produces blood pressure depression and bradycardic effects and induces Akt phosphorylation. Moreover, to investigate the molecular pathogenesis of hypertension, the cardiovascular effects of PI(3,4,5)P3 in NTS are compared between spontaneous hypertensive rats (SHR) and the normotensive control Wistar-kyoto rats (WKY). Microinjection of 1 mM PI(3,4,5)P3 into the NTS in WKY produces long term blood pressure depression compared to SHR. In 16 weeks old WKY, PI(3,4,5)P3 also produces the greater bradycardic effects than in 16 weeks old SHR. Taken together, these results suggested that PI(3,4,5)P3 activated Akt signaling in NTS may play a role in central cardiovascular regulation. Moreover, there may be something defect in PI(3,4,5)P3 downstream in SHR, leading to the hypertention.

Neurogranin (Ng) and neuromodulin (Nm) are neural-specific and Ca2+-sensitive calmodulin (CaM)-binding protein which may be involved in the regulation of Ca2+ dependent NO production. In addition, since the high affinity of Ng and Nm with PI(3,4,5)P3, Ng and Nm may also modulate the NO production induced by PI(3,4,5)P3. The recombinant proteins and specific antibodies of Ng and Nm are prepared for this study. Microinjections of Ng or Nm into the NTS of SD rats do not induce any cardiovascular effects. However, pretreatment of Ng (0.3 �慊/�愮) or Nm (0.3 �慊/�愮) can attenuate the cardiovascular effects of PI(3,4,5)P3. Thus, Ng and Nm may modulate cardiovascular effects of PI(3,4,5)P3 in NTS.

Contents


Contents 1
Abbreviations 2
中文摘要 4
English Abstract 6
Chapter 1
General Introduction
1.1 Backgrounds and Significance 9
1.2 Specific Aims 20
1.3 References 21
Chapter 2
Lipid Second Messenger Activated Akt/PKB Signaling in the Nucleus Tractus Solitarii Contributes to Central Cardiovascular Regulation
2.1 Summary 28
2.2 Introduction 30
2.3 Materials and Methods 34
2.4 Results 41
2.5 Discussion 47
2.6 References 54
Figures and Figure Legends 59
Chapter 3
Roles of Neurogranin and Neuromodulin in Nucleus Tractus Solitarii
3.1 Summary 84
3.2 Introduction 85
3.3 Materials and Methods 89
3.4 Results 101
3.5 Discussion 106
3.6 References 110
Figures and Figure Legends 114
Chapter 4
Future Perspectives
4.1 Future Perspectives 134
4.2 References 138
Appendix 139

Alexander, K. A., Wakim, B. T., Doyle, G. S., Walsh, K. A., and Storm, D. R. (1988). Identification and characterization of the calmodulin-binding domain of neuromodulin, a neurospecific calmodulin-binding protein. J Biol Chem 263, 7544-7549.

Apel, E. D., Byford, M. F., Au, D., Walsh, K. A., and Storm, D. R. (1990). Identification of the protein kinase C phosphorylation site in neuromodulin. Biochemistry 29, 2330-2335.

Barthel, A., Kohn, A. D., Luo, Y., and Roth, R. A. (1997). A constitutively active version of the Ser/Thr kinase Akt induces production of the ob gene product, leptin, in 3T3-L1 adipocytes. Endocrinology 138, 3559-3562.

Barthel, A., Okino, S. T., Liao, J., Nakatani, K., Li, J., Whitlock, J. P., Jr., and Roth, R. A. (1999). Regulation of GLUT1 gene transcription by the serine/threonine kinase Akt1. J Biol Chem 274, 20281-20286.

Baudier, J., Deloulme, J. C., Van Dorsselaer, A., Black, D., and Matthes, H. W. (1991). Purification and characterization of a brain-specific protein kinase C substrate, neurogranin (p17). Identification of a consensus amino acid sequence between neurogranin and neuromodulin (GAP43) that corresponds to the protein kinase C phosphorylation site and the calmodulin-binding domain. J Biol Chem 266, 229-237.

Bevan, P. (2001). Insulin signalling. J Cell Sci 114, 1429-1430.

Cohen, R. W., Margulies, J. E., Coulter, P. M., 2nd, and Watson, J. B. (1993). Functional consequences of expression of the neuron-specific, protein kinase C substrate RC3 (neurogranin) in Xenopus oocytes. Brain Res 627, 147-152.

Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., and Zeiher, A. M. (1999). Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399, 601-605.

Downward, J. (1998). SIGNAL TRANSDUCTION: Lipid-Regulated Kinases: Some Common Themes at Last. Science 279, 673-674.

Erickson, J. T., and Millhorn, D. E. (1994). Hypoxia and electrical stimulation of the carotid sinus nerve induce Fos-like immunoreactivity within catecholaminergic and serotoninergic neurons of the rat brainstem. J Comp Neurol 348, 161-182.

Fedorov, N. B., Pasinelli, P., Oestreicher, A. B., DeGraan, P. N., and Reymann, K. G. (1995). Antibodies to postsynaptic PKC substrate neurogranin prevent long-term potentiation in hippocampal CA1 neurons. Eur J Neurosci 7, 819-822.

Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997). PI3K: downstream AKTion blocks apoptosis. Cell 88, 435-437.

Fruman, D. A., Meyers, R. E., and Cantley, L. C. (1998). Phosphoinositide kinases. Annu Rev Biochem 67, 481-507.

Fulton, D., Gratton, J. P., McCabe, T. J., Fontana, J., Fujio, Y., Walsh, K., Franke, T. F., Papapetropoulos, A., and Sessa, W. C. (1999). Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399, 597-601.

Hayashi, F., Coles, S. K., Bach, K. B., Mitchell, G. S., and McCrimmon, D. R. (1993). Time-dependent phrenic nerve responses to carotid afferent activation: intact vs. decerebellate rats. Am J Physiol 265, R811-R819.

Huang, F. L., Yoshida, Y., Nakabayashi, H., Young, W. S., 3rd, and Huang, K. P. (1988). Immunocytochemical localization of protein kinase C isozymes in rat brain. J Neurosci 8, 4734-4744.

Huang, K. P., Huang, F. L., and Chen, H. C. (1993). Characterization of a 7.5-kDa protein kinase C substrate (RC3 protein, neurogranin) from rat brain. Arch Biochem Biophys 305, 570-580.

Klann, E., Chen, S. J., and Sweatt, J. D. (1992). Increased phosphorylation of a 17-kDa protein kinase C substrate (P17) in long-term potentiation. J Neurochem 58, 1576-1579.

Kobayashi, M., Cheng, Z. B., Tanaka, K., and Nosaka, S. (1999). Is the aortic depressor nerve involved in arterial chemoreflexes in rats? J Auton Nerv Syst 78, 38-48.

Kohn, A. D., Summers, S. A., Birnbaum, M. J., and Roth, R. A. (1996). Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271, 31372-31378.

Krook, A., Roth, R. A., Jiang, X. J., Zierath, J. R., and Wallberg-Henriksson, H. (1998). Insulin-stimulated Akt kinase activity is reduced in skeletal muscle from NIDDM subjects. Diabetes 47, 1281-1286.

Leslie, R. A. (1985). Neuroactive substances in the dorsal vagal complex of the medulla oblongata: nucleus of the tractus solitarius, area postrema and dorsal motor nucleus of the vagus. Neurochem Int 7, 191-212.

Liao, J., Barthel, A., Nakatani, K., and Roth, R. A. (1998). Activation of protein kinase B/Akt is sufficient to repress the glucocorticoid and cAMP induction of phosphoenolpyruvate carboxykinase gene. J Biol Chem 273, 27320-27324.

Lo, W. C., Jan, C. R., Chiang, H. T., and Tseng, C. J. (2000). Modulatory effects of carbon monoxide on baroreflex activation in nucleus tractus solitarii of rats. Hypertension 35, 1253-1257.

Loewy, A. D. (1981). Descending pathways to sympathetic and parasympathetic preganglionic neurons. J Auton Nerv Syst 3, 265-275.

Martzen, M. R., and Slemmon, J. R. (1995). The dendritic peptide neurogranin can regulate a calmodulin-dependent target. J Neurochem 64, 92-100.

Miao, H. H., Ye, J. S., Wong, S. L., Wang, B. X., Li, X. Y., and Sheu, F. S. (2000). Oxidative modification of neurogranin by nitric oxide: an amperometric study. Bioelectrochemistry 51, 163-173.

Morbidelli, L., Donnini, S., and Ziche, M. (2003). Role of nitric oxide in the modulation of angiogenesis. Curr Pharm Des 9, 521-530.

Mosqueda-Garcia, R., Tseng, C. J., Appalsamy, M., Beck, C., and Robertson, D. (1991). Cardiovascular excitatory effects of adenosine in the nucleus of the solitary tract. Hypertension 18, 494-502.

Mosqueda-Garcia, R., Tseng, C. J., Appalsamy, M., and Robertson, D. (1990). Cardiovascular effects of microinjection of angiotensin II in the brainstem of renal hypertensive rats. J Pharmacol Exp Ther 255, 374-381.

Numao, Y., Siato, M., Terui, N., and Kumada, M. (1985). The aortic nerve-sympathetic reflex in the rat. J Auton Nerv Syst 13, 65-79.

Ohta, H., and Talman, W. T. (1995). Alteration of baroreceptor and chemoreceptor reflexes in spontaneously hypertensive rats. Clin Exp Pharmacol Physiol Suppl 22, S60-S61.

Prichard, L., Deloulme, J. C., and Storm, D. R. (1999). Interactions between neurogranin and calmodulin in vivo. J Biol Chem 274, 7689-7694.

Ramakers, G. M., De Graan, P. N., Urban, I. J., Kraay, D., Tang, T., Pasinelli, P., Oestreicher, A. B., and Gispen, W. H. (1995). Temporal differences in the phosphorylation state of pre- and postsynaptic protein kinase C substrates B-50/GAP-43 and neurogranin during long-term potentiation. J Biol Chem 270, 13892-13898.

Ramakers, G. M., Gerendasy, D. D., and de Graan, P. N. (1999). Substrate phosphorylation in the protein kinase Cgamma knockout mouse. J Biol Chem 274, 1873-1874.

Rondinone, C. M., Carvalho, E., Wesslau, C., and Smith, U. P. (1999). Impaired glucose transport and protein kinase B activation by insulin, but not okadaic acid, in adipocytes from subjects with Type II diabetes mellitus. Diabetologia 42, 819-825.

Sapru, H. N., Gonzalez, E., and Krieger, A. J. (1981). Aortic nerve stimulation in the rat: cardiovascular and respiratory responses. Brain Res Bull 6, 393-398.

Sapru, H. N., and Krieger, A. J. (1977). Carotid and aortic chemoreceptor function in the rat. J Appl Physiol 42, 344-348.

Sato, T., Xiao, D. M., Li, H., Huang, F. L., and Huang, K. P. (1995). Structure and regulation of the gene encoding the neuron-specific protein kinase C substrate neurogranin (RC3 protein). J Biol Chem 270, 10314-10322.

Shepherd, P. R., Withers, D. J., and Siddle, K. (1998). Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling. Biochem J 333, 471-490.

Sheu, F. S., Mahoney, C. W., Seki, K., and Huang, K. P. (1996). Nitric oxide modification of rat brain neurogranin affects its phosphorylation by protein kinase C and affinity for calmodulin. J Biol Chem 271, 22407-22413.

Sun, M. K. (1995). Central neural organization and control of sympathetic nervous system in mammals. Prog Neurobiol 47, 157-233.

Tseng, C. J., Biaggioni, I., Appalsamy, M., and Robertson, D. (1988). Purinergic receptors in the brainstem mediate hypotension and bradycardia. Hypertension 11, 191-197.

Tseng, C. J., Appalsamy, M., Robertson, D., and Mosqueda-Garcia, R. (1993). Effects of nicotine on brain stem mechanisms of cardiovascular control. J Pharmacol Exp Ther 265, 1511-1518.

Tseng, C. J., Chou, L. L., Ger, L. P., and Tung, C. S. (1994). Cardiovascular effects of angiotensin III in brainstem nuclei of normotensive and hypertensive rats. J Pharmacol Exp Ther 268, 558-564.

Tseng, C. J., Ger, L. P., and Tung, C. S. (1991). Interrelation between alpha 2-adrenoreceptor system and neuropeptide Y in rat nucleus tractus solitarii. Proc Natl Sci Counc Repub China B 15, 86-91.

Tseng, C. J., Liu, H. Y., Lin, H. C., Ger, L. P., Tung, C. S., and Yen, M. H. (1996). Cardiovascular effects of nitric oxide in the brain stem nuclei of rats. Hypertension 27, 36-42.

Tseng, C. J., Mosqueda-Garcia, R., Appalsamy, M., and Robertson, D. (1989). Cardiovascular effects of neuropeptide Y in rat brainstem nuclei. Circ Res 64, 55-61.

Vanhaesebroeck, B., and Waterfield, M. D. (1999). Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res 253, 239-254.

Walker, K. S., Deak, M., Paterson, A., Hudson, K., Cohen, P., and Alessi, D. R. (1998). Activation of protein kinase B beta and gamma isoforms by insulin in vivo and by 3-phosphoinositide-dependent protein kinase-1 in vitro: comparison with protein kinase B alpha. Biochem J 331, 299-308.

Wang, D., and Sul, H. S. (1998). Insulin stimulation of the fatty acid synthase promoter is mediated by the phosphatidylinositol 3-kinase pathway. Involvement of protein kinase B/Akt. J Biol Chem 273, 25420-25426.

Watson, J. B., Battenberg, E. F., Wong, K. K., Bloom, F. E., and Sutcliffe, J. G. (1990). Subtractive cDNA cloning of RC3, a rodent cortex-enriched mRNA encoding a novel 78 residue protein. J Neurosci Res 26, 397-408.

Alessi, D. R., James, S. R., Downes, C. P., Holmes, A. B., Gaffney, P. R., Reese, C. B., and Cohen, P. (1997). Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 7, 261-269.

Anggard, E. (1994). Nitric oxide: mediator, murderer, and medicine. Lancet 343, 1199-1206.

Bellacosa, A., Chan, T. O., Ahmed, N. N., Datta, K., Malstrom, S., Stokoe, D., McCormick, F., Feng, J., and Tsichlis, P. (1998). Akt activation by growth factors is a multiple-step process: the role of the PH domain. Oncogene 17, 313-325.

Brown, R. A., Ho, L. K., Weber-Hall, S. J., Shipley, J. M., and Fry, M. J. (1997). Identification and cDNA cloning of a novel mammalian C2 domain-containing phosphoinositide 3-kinase, HsC2-PI3K. Biochem Biophys Res Commun 233, 537-544.

Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M., and Hemmings, B. A. (1995). Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378, 785-789.

Dimmeler, S., Fleming, I., Fisslthaler, B., Hermann, C., Busse, R., and Zeiher, A. M. (1999). Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399, 601-605.

Forstermann, U., Closs, E. I., Pollock, J. S., Nakane, M., Schwarz, P., Gath, I., and Kleinert, H. (1994). Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 23, 1121-1131.

Fruman, D. A., Meyers, R. E., and Cantley, L. C. (1998). Phosphoinositide kinases. Annu Rev Biochem 67, 481-507.

Fulton, D., Gratton, J. P., McCabe, T. J., Fontana, J., Fujio, Y., Walsh, K., Franke, T. F., Papapetropoulos, A., and Sessa, W. C. (1999). Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399, 597-601.

Ito, K., Hirooka, Y., Sakai, K., Kishi, T., Kaibuchi, K., Shimokawa, H., and Takeshita, A. (2003). Rho/Rho-kinase pathway in brain stem contributes to blood pressure regulation via sympathetic nervous system: possible involvement in neural mechanisms of hypertension. Circ Res 92, 1337-1343.

Jiang, T., Sweeney, G., Rudolf, M. T., Klip, A., Traynor-Kaplan, A., and Tsien, R. Y. (1998). Membrane-permeant esters of phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 273, 11017-11024.

Kane, L. P., Lin, J., and Weiss, A. (2000). Signal transduction by the TCR for antigen. Curr Opin Immunol 12, 242-249.

Katso, R., Okkenhaug, K., Ahmadi, K., White, S., Timms, J., and Waterfield, M. D. (2001). Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol 17, 615-675.

Laufs, U., and Liao, J. K. (1998). Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem 273, 24266-24271.

Leslie, R. A. (1985). Neuroactive substances in the dorsal vagal complex of the medulla oblongata: nucleus of the tractus solitarius, area postrema and dorsal motor nucleus of the vagus. Neurochem Int 7, 191-212.

Lin, H. C., Wan, F. J., Kang, B. H., Wu, C. C., and Tseng, C. J. (1999). Systemic administration of lipopolysaccharide induces release of nitric oxide and glutamate and c-fos expression in the nucleus tractus solitarii of rats. Hypertension 33, 1218-1224.

Lo, W. C., Jan, C. R., Chiang, H. T., and Tseng, C. J. (2000). Modulatory effects of carbon monoxide on baroreflex activation in nucleus tractus solitarii of rats. Hypertension 35, 1253-1257.

Lo, W. C., Lin, H. C., Ger, L. P., Tung, C. S., and Tseng, C. J. (1997). Cardiovascular effects of nitric oxide and N-methyl-D-aspartate receptors in the nucleus tractus solitarii of rats. Hypertension 30, 1499-1503.

Ming, X. F., Viswambharan, H., Barandier, C., Ruffieux, J., Kaibuchi, K., Rusconi, S., and Yang, Z. (2002). Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells. Mol Cell Biol 22, 8467-8477.

Montagnani, M., Chen, H., Barr, V. A., and Quon, M. J. (2001). Insulin-stimulated activation of eNOS is independent of Ca2+ but requires phosphorylation by Akt at Ser(1179). J Biol Chem 276, 30392-30398.

Montagnani, M., Ravichandran, L. V., Chen, H., Esposito, D. L., and Quon, M. J. (2002). Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells. Mol Endocrinol 16, 1931-1942.

Mosqueda-Garcia, R., Tseng, C., Appalsamy, M., Beck, C., and Robertson, D. (1991). Cardiovascular excitatory effects of adenosine in the nucleus of the solitary tract. Hypertension 18, 494-502.

Mosqueda-Garcia, R., Tseng, C. J., Appalsamy, M., and Robertson, D. (1990). Cardiovascular effects of microinjection of angiotensin II in the brainstem of renal hypertensive rats. J Pharmacol Exp Ther 255, 374-381.

Nystrom, F. H., and Quon, M. J. (1999). Insulin signalling: metabolic pathways and mechanisms for specificity. Cell Signal 11, 563-574.

Paton, J. F., Deuchars, J., Ahmad, Z., Wong, L. F., Murphy, D., and Kasparov, S. (2001). Adenoviral vector demonstrates that angiotensin II-induced depression of the cardiac baroreflex is mediated by endothelial nitric oxide synthase in the nucleus tractus solitarii of the rat. J Physiol 531, 445-458.

Peunova, N., and Enikolopov, G. (1995). Nitric oxide triggers a switch to growth arrest during differentiation of neuronal cells. Nature 375, 68-73.

Romashkova, J. A., and Makarov, S. S. (1999). NF-kappaB is a target of AKT in anti-apoptotic PDGF signalling. Nature 401, 86-90.

Rommel, C., Clarke, B. A., Zimmermann, S., Nunez, L., Rossman, R., Reid, K., Moelling, K., Yancopoulos, G. D., and Glass, D. J. (1999). Differentiation stage-specific inhibition of the Raf-MEK-ERK pathway by Akt. Science 286, 1738-1741.

Secondo, A., Sirabella, R., Formisano, L., D''Alessio, A., Castaldo, P., Amoroso, S., Ingleton, P., Di Renzo, G., and Annunziato, L. (2003). Involvement of PI3''-K, mitogen-activated protein kinase and protein kinase B in the up-regulation of the expression of nNOSalpha and nNOSbeta splicing variants induced by PRL-receptor activation in GH3 cells. J Neurochem 84, 1367-1377.

Smith, L. K., Vlahos, C. J., Reddy, K. K., Falck, J. R., and Garner, C. W. (1995). Wortmannin and LY294002 inhibit the insulin-induced down-regulation of IRS-1 in 3T3-L1 adipocytes. Mol Cell Endocrinol 113, 73-81.

Sparrow, J. R. (1994). Inducible nitric oxide synthase in the central nervous system. J Mol Neurosci 5, 219-229.

Stephens, L., Anderson, K., Stokoe, D., Erdjument-Bromage, H., Painter, G. F., Holmes, A. B., Gaffney, P. R., Reese, C. B., McCormick, F., Tempst, P., et al. (1998). Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. Science 279, 710-714.

Stephens, L., Cooke, F. T., Walters, R., Jackson, T., Volinia, S., Gout, I., Waterfield, M. D., and Hawkins, P. T. (1994). Characterization of a phosphatidylinositol-specific phosphoinositide 3-kinase from mammalian cells. Curr Biol 4, 203-214.

Stephens, L., Hawkins, P. T., Eguinoa, A., and Cooke, F. (1996). A heterotrimeric GTPase-regulated isoform of PI3K and the regulation of its potential effectors. Philos Trans R Soc Lond B Biol Sci 351, 211-215.

Tang, X., and Downes, C. P. (1997). Purification and characterization of Gbetagamma-responsive phosphoinositide 3-kinases from pig platelet cytosol. J Biol Chem 272, 14193-14199.

Tseng, C. J., Biaggioni, I., Appalsamy, M., and Robertson, D. (1988). Purinergic receptors in the brainstem mediate hypotension and bradycardia. Hypertension 11, 191-197.

Tseng, C. J., Appalsamy, M., Robertson, D., and Mosqueda-Garcia, R. (1993). Effects of nicotine on brain stem mechanisms of cardiovascular control. J Pharmacol Exp Ther 265, 1511-1518.

Tseng, C. J., Chou, L. L., Ger, L. P., and Tung, C. S. (1994). Cardiovascular effects of angiotensin III in brainstem nuclei of normotensive and hypertensive rats. J Pharmacol Exp Ther 268, 558-564.

Tseng, C. J., Ger, L. P., and Tung, C. S. (1991). Interrelation between alpha 2-adrenoreceptor system and neuropeptide Y in rat nucleus tractus solitarii. Proc Natl Sci Counc Repub China B 15, 86-91.

Tseng, C. J., Liu, H. Y., Lin, H. C., Ger, L. P., Tung, C. S., and Yen, M. H. (1996). Cardiovascular effects of nitric oxide in the brain stem nuclei of rats. Hypertension 27, 36-42.

Tseng, C. J., Mosqueda-Garcia, R., Appalsamy, M., and Robertson, D. (1989). Cardiovascular effects of neuropeptide Y in rat brainstem nuclei. Circ Res 64, 55-61.

Vanhaesebroeck, B., Leevers, S. J., Ahmadi, K., Timms, J., Katso, R., Driscoll, P. C., Woscholski, R., Parker, P. J., and Waterfield, M. D. (2001). Synthesis and function of 3-phosphorylated inositol lipids. Annu Rev Biochem 70, 535-602.

Vanhaesebroeck, B., and Waterfield, M. D. (1999). Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res 253, 239-254.

Vincent, S. R. (1994). Nitric oxide: a radical neurotransmitter in the central nervous system. Prog Neurobiol 42, 129-160.

Zeng, G., Nystrom, F. H., Ravichandran, L. V., Cong, L. N., Kirby, M., Mostowski, H., and Quon, M. J. (2000). Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation 101, 1539-1545.

Zeng, G., and Quon, M. J. (1996). Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells. J Clin Invest 98, 894-898.

Alvarez-Bolado, G., Rodriguez-Sanchez, P., Tejero-Diez, P., Fairen, A., and Diez-Guerra, F. J. (1996). Neurogranin in the development of the rat telencephalon. Neuroscience 73, 565-580.

Baudier, J., Deloulme, J. C., Van Dorsselaer, A., Black, D., and Matthes, H. W. (1991). Purification and characterization of a brain-specific protein kinase C substrate, neurogranin (p17). Identification of a consensus amino acid sequence between neurogranin and neuromodulin (GAP43) that corresponds to the protein kinase C phosphorylation site and the calmodulin-binding domain. J Biol Chem 266, 229-237.

Fedorov, N. B., Pasinelli, P., Oestreicher, A. B., DeGraan, P. N., and Reymann, K. G. (1995). Antibodies to postsynaptic PKC substrate neurogranin prevent long-term potentiation in hippocampal CA1 neurons. Eur J Neurosci 7, 819-822.

Gerendasy, D. D., and Sutcliffe, J. G. (1997). RC3/neurogranin, a postsynaptic calpacitin for setting the response threshold to calcium influxes. Mol Neurobiol 15, 131-163.

Higo, N., Oishi, T., Yamashita, A., Matsuda, K., and Hayashi, M. (2003). Cell type- and region-specific expression of protein kinase C-substrate mRNAs in the cerebellum of the macaque monkey. J Comp Neurol 467, 135-149.

Hoffman, P. N. (1989). Expression of GAP-43, a rapidly transported growth-associated protein, and class II beta tubulin, a slowly transported cytoskeletal protein, are coordinated in regenerating neurons. J Neurosci 9, 893-897.

Huang, F. L., Yoshida, Y., Nakabayashi, H., Young, W. S., 3rd, and Huang, K. P. (1988). Immunocytochemical localization of protein kinase C isozymes in rat brain. J Neurosci 8, 4734-4744.

Huang, K. P., Huang, F. L., and Chen, H. C. (1993). Characterization of a 7.5-kDa protein kinase C substrate (RC3 protein, neurogranin) from rat brain. Arch Biochem Biophys 305, 570-580.

Jacobson, R. D., Virag, I., and Skene, J. H. (1986). A protein associated with axon growth, GAP-43, is widely distributed and developmentally regulated in rat CNS. J Neurosci 6, 1843-1855.

Klann, E., Chen, S. J., and Sweatt, J. D. (1993). Mechanism of protein kinase C activation during the induction and maintenance of long-term potentiation probed using a selective peptide substrate. Proc Natl Acad Sci U S A 90, 8337-8341.

Li, J., Pak, J. H., Huang, F. L., and Huang, K. P. (1999). N-methyl-D-aspartate induces neurogranin/RC3 oxidation in rat brain slices. J Biol Chem 274, 1294-1300.

Liu, Y. C., and Storm, D. R. (1990). Regulation of free calmodulin levels by neuromodulin: neuron growth and regeneration. Trends Pharmacol Sci 11, 107-111.

Lo, W. C., Jan, C. R., Chiang, H. T., and Tseng, C. J. (2000). Modulatory effects of carbon monoxide on baroreflex activation in nucleus tractus solitarii of rats. Hypertension 35, 1253-1257.

Lo, W. C., Lin, H. C., Ger, L. P., Tung, C. S., and Tseng, C. J. (1997). Cardiovascular effects of nitric oxide and N-methyl-D-aspartate receptors in the nucleus tractus solitarii of rats. Hypertension 30, 1499-1503.

Lu, P. J., and Chen, C. S. (1997). Selective recognition of phosphatidylinositol 3,4,5-trisphosphate by a synthetic peptide. J Biol Chem 272, 466-472.

Mahoney, C. W., Pak, J. H., and Huang, K. P. (1996). Nitric oxide modification of rat brain neurogranin. Identification of the cysteine residues involved in intramolecular disulfide bridge formation using site-directed mutagenesis. J Biol Chem 271, 28798-28804.

Meiri, K. F., Willard, M., and Johnson, M. I. (1988). Distribution and phosphorylation of the growth-associated protein GAP-43 in regenerating sympathetic neurons in culture. J Neurosci 8, 2571-2581.

Miao, H. H., Ye, J. S., Wong, S. L., Wang, B. X., Li, X. Y., and Sheu, F. S. (2000). Oxidative modification of neurogranin by nitric oxide: an amperometric study. Bioelectrochemistry 51, 163-173.

Neuner-Jehle, M., Denizot, J. P., and Mallet, J. (1996). Neurogranin is locally concentrated in rat cortical and hippocampal neurons. Brain Res 733, 149-154.

Nishizuka, Y. (1992). Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258, 607-614.

Pak, J. H., Huang, F. L., Li, J., Balschun, D., Reymann, K. G., Chiang, C., Westphal, H., and Huang, K. P. (2000). Involvement of neurogranin in the modulation of calcium/calmodulin-dependent protein kinase II, synaptic plasticity, and spatial learning: a study with knockout mice. Proc Natl Acad Sci U S A 97, 11232-11237.

Ramakers, G. M., De Graan, P. N., Urban, I. J., Kraay, D., Tang, T., Pasinelli, P., Oestreicher, A. B., and Gispen, W. H. (1995). Temporal differences in the phosphorylation state of pre- and postsynaptic protein kinase C substrates B-50/GAP-43 and neurogranin during long-term potentiation. J Biol Chem 270, 13892-13898.

Ramakers, G. M., Gerendasy, D. D., and de Graan, P. N. (1999). Substrate phosphorylation in the protein kinase Cgamma knockout mouse. J Biol Chem 274, 1873-1874.

Ramakers, G. M., Heinen, K., Gispen, W. H., and de Graan, P. N. (2000a). Long term depression in the CA1 field is associated with a transient decrease in pre- and postsynaptic PKC substrate phosphorylation. J Biol Chem 275, 28682-28687.

Ramakers, G. M., Pasinelli, P., van Beest, M., van der Slot, A., Gispen, W. H., and De Graan, P. N. (2000b). Activation of pre- and postsynaptic protein kinase C during tetraethylammonium-induced long-term potentiation in the CA1 field of the hippocampus. Neurosci Lett 286, 53-56.

Represa, A., Deloulme, J. C., Sensenbrenner, M., Ben-Ari, Y., and Baudier, J. (1990). Neurogranin: immunocytochemical localization of a brain-specific protein kinase C substrate. J Neurosci 10, 3782-3792.

Saitoh, T., Horsburgh, K., and Masliah, E. (1993). Hyperactivation of signal transduction systems in Alzheimer''s disease. Ann N Y Acad Sci 695, 34-41.

Sato, T., Xiao, D. M., Li, H., Huang, F. L., and Huang, K. P. (1995). Structure and regulation of the gene encoding the neuron-specific protein kinase C substrate neurogranin (RC3 protein). J Biol Chem 270, 10314-10322.

Schneider, J. C., El Kebir, D., Chereau, C., Lanone, S., Huang, X. L., De Buys Roessingh, A. S., Mercier, J. C., Dall''Ava-Santucci, J., and Dinh-Xuan, A. T. (2003). Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation. Am J Physiol Heart Circ Physiol 284, H2311-2319.

Sheu, F. S., Mahoney, C. W., Seki, K., and Huang, K. P. (1996). Nitric oxide modification of rat brain neurogranin affects its phosphorylation by protein kinase C and affinity for calmodulin. J Biol Chem 271, 22407-22413.

Singec, I., Knoth, R., Ditter, M., Frotscher, M., and Volk, B. (2003). Neurogranin expression by cerebellar neurons in rodents and non-human primates. J Comp Neurol 459, 278-289.

Watson, J. B., Battenberg, E. F., Wong, K. K., Bloom, F. E., and Sutcliffe, J. G. (1990). Subtractive cDNA cloning of RC3, a rodent cortex-enriched mRNA encoding a novel 78 residue protein. J Neurosci Res 26, 397-408.

Watson, J. B., Sutcliffe, J. G., and Fisher, R. S. (1992). Localization of the protein kinase C phosphorylation/calmodulin-binding substrate RC3 in dendritic spines of neostriatal neurons. Proc Natl Acad Sci U S A 89, 8581-8585.

Watson, J. B., Szijan, I., and Coulter, P. M., 2nd (1994). Localization of RC3 (neurogranin) in rat brain subcellular fractions. Brain Res Mol Brain Res 27, 323-328.

Wu, J., Wang, Y., Rowan, M. J., and Anwyl, R. (1997). Evidence for involvement of the neuronal isoform of nitric oxide synthase during induction of long-term potentiation and long-term depression in the rat dentate gyrus in vitro. Neuroscience 78, 393-398.

Higo, N., Oishi, T., Yamashita, A., Matsuda, K., and Hayashi, M. (2003). Cell type- and region-specific expression of protein kinase C-substrate mRNAs in the cerebellum of the macaque monkey. J Comp Neurol 467, 135-149.

Ito, K., Hirooka, Y., Sakai, K., Kishi, T., Kaibuchi, K., Shimokawa, H., and Takeshita, A. (2003). Rho/Rho-kinase pathway in brain stem contributes to blood pressure regulation via sympathetic nervous system: possible involvement in neural mechanisms of hypertension. Circ Res 92, 1337-1343.

Ming, X. F., Viswambharan, H., Barandier, C., Ruffieux, J., Kaibuchi, K., Rusconi, S., and Yang, Z. (2002). Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells. Mol Cell Biol 22, 8467-8477.

Montagnani, M., Chen, H., Barr, V. A., and Quon, M. J. (2001). Insulin-stimulated activation of eNOS is independent of Ca2+ but requires phosphorylation by Akt at Ser(1179). J Biol Chem 276, 30392-30398.

Ramakers, G. M., De Graan, P. N., Urban, I. J., Kraay, D., Tang, T., Pasinelli, P., Oestreicher, A. B., and Gispen, W. H. (1995). Temporal differences in the phosphorylation state of pre- and postsynaptic protein kinase C substrates B-50/GAP-43 and neurogranin during long-term potentiation. J Biol Chem 270, 13892-13898.

Ramakers, G. M., Heinen, K., Gispen, W. H., and de Graan, P. N. (2000a). Long term depression in the CA1 field is associated with a transient decrease in pre- and postsynaptic PKC substrate phosphorylation. J Biol Chem 275, 28682-28687.

Ramakers, G. M., Pasinelli, P., van Beest, M., van der Slot, A., Gispen, W. H., and De Graan, P. N. (2000b). Activation of pre- and postsynaptic protein kinase C during tetraethylammonium-induced long-term potentiation in the CA1 field of the hippocampus. Neurosci Lett 286, 53-56.