alpha-melanocyte-stimulating hormone (alpha-MSH) 在食慾、代謝速率與免疫方面被認為是個重要的調節因子。最近更有文獻指出，alpha-MSH也會影響交感神經的活性與血壓的調控。在本研究中，我們利用微量注射的方式將alpha-MSH注射入自發性高血壓老鼠 (spontaneously hypertensive rats，SHR) 的孤立束核 (nucleus the tractus solitarii，NTS) 中，探討alpha-MSH所產生的心血管調控作用；同時，由於目前已知一氧化氮和心血管調控有密切的關聯，因此我們也將更進一步探討alpha-MSH對心血管調控產生的作用是否與一氧化氮的調控機制相關。在麻醉狀態下的自發性高血壓老鼠，利用單邊微量注射alpha-MSH (0.3~300 pmol) 到孤立束核內會引起血壓下降與心跳的減緩，且呈現顯著的劑量效應；而此降血壓與減緩心跳的作用會被melanocortin 3/4-receptor的拮抗劑，SHU9119，抑制。注射一氧化氮的前趨物L-arginine做前處理，能使得alpha-MSH所產生的降血壓與減緩心跳的作用時間增長；相反地，一氧化氮合成酶的抑制劑L-NAME (N-nitro-L-arginine methyl ester)，則會抑制alpha-MSH產生的降血壓作用。接著利用誘導性一氧化氮合成酶 (inducible nitric oxide synthase) 的抑制劑aminoguanidine (AG)，可明顯觀察出AG能抑制alpha-MSH在心血管調控上的作用；而神經性一氧化氮合成酶 (neuronal nitric oxide synthase) 抑制劑7-nitroindazole，則無此效應。因此我們推測注射alpha-MSH於自發性高血壓老鼠的孤立束核中具有降血壓與減緩心跳的作用，而此機制似乎是藉由melanocortin 4-receptor訊息機轉進而活化一氧化氮合成酶，使其進而在孤立束核產生一氧化氮而達到心血管調控作用。

alpha-melanocyte stimulating hormone (alpha-MSH) is an important regulator of food intake, metabolic rate, and inflammation. Recently, alpha-MSH was shown to influence sympathetic activity and blood pressure regulation. In the present study, we investigated the cardiovascular effects of alpha-MSH in the nucleus tractus solitarii (NTS) of spontaneously hypertensive rats (SHR). Because nitric oxide (NO) is well-known to involve in central cardiovascular regulation, we elucidated the role of NO in the cardiovascular responses induced by alpha-MSH. In urethane-anesthetized SHR, unilateral microinjection of alpha-MSH (0.3-300 pmol) into the NTS produced dose-responsive depressor and bradycardic effects. The cardiovascular effects of alpha-MSH were abrogated by the antagonist of melanocortin receptor (MC3/4-R), SHU9119. Pretreatment with precursor of nitric oxide, L-arginine, enhanced the duration of alpha-MSH-mediated hypotensive effects, whereas prior application of L-NAME, a universal inhibitor of nitric oxide synthase (NOS), significantly attenuated the effects of alpha-MSH. Prior injection with inhibitor of inducible NOS, aminoguanidine, but not inhibitor of neuronal NOS, 7-nitroindazole, attenuated the hypotensive effect of alpha-MSH. In summary, these results indicated alpha-MSH induced depressor and bradycardic effects in the NTS of SHR. Besides, the hypotensive mechanism of alpha-MSH was mediated via MC4-R and involved with iNOS activation in the NTS of SHR.

Pages
Contents ⅰ
Abstract in Chinese iv
Abstract in English vi
Abbreviations vii

Introduction
Pro-opiomelanocortin (POMC)………………………………1
POMC-derived neuropeptides………………………………2
Melanocortin receptors…………………………………………………2
alpha-melanocyte-stimulating hormone (alpha-MSH)………3
Nitric oxide (NO)………………………………………………4
Cardiovascular effects of NO in the NTS…………………6
Hypertensive animal models…………………………………8
Specific aims……………………………………………………9

Methods and Materials
Animals...............................................10
NTS microinjection....................................10
Cell cultures.........................................13
Western blot analysis.................................13
Immunohistochemical staining..........................14
Statistical analysis..................................15

Results
Microinjection of alpha-MSH into the NTS caused dose-responsive hypotension in SHR and WKY rats..........16
Antagonist of MC3/4-R, SHU9119, inhibited the cardio-vascular effects of alpha-MSH effectively in SHR and WKY rats................................................16
NO pathway participated in alpha-MSH-induced cardio-vascular effects in SHR and WKY rats..................17
iNOS is involved in the cardiovascular effects of alpha-MSH................................................18
PKA inhibitor, H89, abolished the cardiovascular effects of alpha-MSH in SHR................................19
iNOS was co-localized with MC4-R in the NTS of SHR and WKY rats................................................19

Discussion..........................................20

Future Perspectives.................................24

References..........................................26

Tables...............................................34

Figures..............................................36

Aitman, T. J., Glazier, A. M., Wallace, C. A., Cooper, L. D., Norsworthy, P. J., Wahid, F. N., Al-Majali, K. M., Trembling, P. M., Mann, C. J., Shoulders, C. C., et al. (1999). Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats. Nat Genet 21, 76-83.
Aktan, F. (2004). iNOS-mediated nitric oxide production and its regulation. Life Sci 75, 639-653.
Anggard, E. (1994). Nitric oxide: mediator, murderer, and medicine. Lancet 343, 1199-1206.
Bredt, D. S., and Snyder, S. H. (1992). Nitric oxide, a novel neuronal messenger. Neuron 8, 3-11.
Butler, A. A., Marks, D. L., Fan, W., Kuhn, C. M., Bartolome, M., and Cone, R. D. (2001). Melanocortin-4 receptor is required for acute homeostatic responses to increased dietary fat. Nat Neurosci 4, 605-611.
Carter, D. A., and Lightman, S. L. (1987). alpha-MSH exhibits opioid antagonist-like effects in the brainstem of rats. Peptides 8, 1173-1175.
Catania, A., Gatti, S., Colombo, G., and Lipton, J. M. (2004). Targeting melanocortin receptors as a novel strategy to control inflammation. Pharmacol Rev 56, 1-29.
Chianca, D. A., Jr., Lin, L. H., Dragon, D. N., and Talman, W. T. (2004). NMDA receptors in nucleus tractus solitarii are linked to soluble guanylate cyclase. Am J Physiol Heart Circ Physiol 286, H1521-1527.
Chowdhary, S., and Townend, J. N. (1999). Role of nitric oxide in the regulation of cardiovascular autonomic control. Clin Sci (Lond) 97, 5-17.
Chretien, M., Benjannet, S., Gossard, F., Gianoulakis, C., Crine, P., Lis, M., and Seidah, N. G. (1979). From beta-lipotropin to beta-endorphin and 'pro-opio-melanocortin'. Can J Biochem 57, 1111-1121.
Ciriello, J. (1983). Brainstem projections of aortic baroreceptor afferent fibers in the rat. Neurosci Lett 36, 37-42.
Ciriello, J., Hrycyshyn, A. W., and Calaresu, F. R. (1981). Glossopharyngeal and vagal afferent projections to the brain stem of the cat: a horseradish peroxidase study. J Auton Nerv Syst 4, 63-79.
Colombari, E., Sato, M. A., Cravo, S. L., Bergamaschi, C. T., Campos, R. R., Jr., and Lopes, O. U. (2001). Role of the medulla oblongata in hypertension. Hypertension 38, 549-554.
Cone, R. D. (1999). The Central Melanocortin System and Energy Homeostasis. Trends Endocrinol Metab 10, 211-216.
Daniels, D., Patten, C. S., Roth, J. D., Yee, D. K., and Fluharty, S. J. (2003). Melanocortin receptor signaling through mitogen-activated protein kinase in vitro and in rat hypothalamus. Brain Res 986, 1-11.
Dunbar, J. C., and Lu, H. (2000). Proopiomelanocortin (POMC) products in the central regulation of sympathetic and cardiovascular dynamics: studies on melanocortin and opioid interactions. Peptides 21, 211-217.
Eberle, A., Kriwaczek, V. M., and Schwyzer, R. (1978). Mechanism of alpha-melanotropin action. Bull Schweiz Akad Med Wiss 34, 99-111.
Fong, A. Y., Talman, W. T., and Lawrence, A. J. (2000). Axonal transport of NADPH-diaphorase and [(3)H]nitro-L-arginine binding, but not [(3)H]cGMP binding, by the rat vagus nerve. Brain Res 878, 240-246.
Gantz, I., Miwa, H., Konda, Y., Shimoto, Y., Tashiro, T., Watson, S. J., DelValle, J., and Yamada, T. (1993). Molecular cloning, expression, and gene localization of a fourth melanocortin receptor. J Biol Chem 268, 15174-15179.
Getting, S. J. (2002). Melanocortin peptides and their receptors: new targets for anti-inflammatory therapy. Trends Pharmacol Sci 23, 447-449.
Gorren, A. C., and Mayer, B. (1998). The versatile and complex enzymology of nitric oxide synthase. Biochemistry (Mosc) 63, 734-743.
Grieco, P., Rossi, C., Colombo, G., Gatti, S., Novellino, E., Lipton, J. M., and Catania, A. (2003). Novel alpha-melanocyte stimulating hormone peptide analogues with high candidacidal activity. J Med Chem 46, 850-855.
Hemmens, B., and Mayer, B. (1998). Enzymology of nitric oxide synthases. Methods Mol Biol 100, 1-32.
Ignarro, L. J., Byrns, R. E., Buga, G. M., and Wood, K. S. (1987). Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ Res 61, 866-879.
Kalia, M., and Mesulam, M. M. (1980). Brain stem projections of sensory and motor components of the vagus complex in the cat: I. The cervical vagus and nodose ganglion. J Comp Neurol 193, 435-465.
Kishi, T., Hirooka, Y., Ito, K., Sakai, K., Shimokawa, H., and Takeshita, A. (2002). Cardiovascular effects of overexpression of endothelial nitric oxide synthase in the rostral ventrolateral medulla in stroke-prone spontaneously hypertensive rats. Hypertension 39, 264-268.
Kishi, T., Hirooka, Y., Kimura, Y., Sakai, K., Ito, K., Shimokawa, H., and Takeshita, A. (2003). Overexpression of eNOS in RVLM improves impaired baroreflex control of heart rate in SHRSP. Rostral ventrolateral medulla. Stroke-prone spontaneously hypertensive rats. Hypertension 41, 255-260.
Krude, H., and Gruters, A. (2000). Implications of proopiomelanocortin (POMC) mutations in humans: the POMC deficiency syndrome. Trends Endocrinol Metab 11, 15-22.
Kuo, J. J., Silva, A. A., and Hall, J. E. (2003). Hypothalamic melanocortin receptors and chronic regulation of arterial pressure and renal function. Hypertension 41, 768-774.
Lerner, A. B. (1993). The discovery of the melanotropins. A history of pituitary endocrinology. Ann N Y Acad Sci 680, 1-12.
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.
Li, S. J., Varga, K., Archer, P., Hruby, V. J., Sharma, S. D., Kesterson, R. A., Cone, R. D., and Kunos, G. (1996). Melanocortin antagonists define two distinct pathways of cardiovascular control by alpha- and gamma-melanocyte-stimulating hormones. J Neurosci 16, 5182-5188.
Lin, L. H., Cassell, M. D., Sandra, A., and Talman, W. T. (1998). Direct evidence for nitric oxide synthase in vagal afferents to the nucleus tractus solitarii. Neuroscience 84, 549-558.
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.
Moncada, S., Palmer, R. M., and Higgs, E. A. (1991). Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43, 109-142.
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.
Mountjoy, K. G., Mortrud, M. T., Low, M. J., Simerly, R. B., and Cone, R. D. (1994). Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain. Mol Endocrinol 8, 1298-1308.
Myers, M. M., Brunelli, S. A., Shair, H. N., Squire, J. M., and Hofer, M. A. (1989). Relationships between maternal behavior of SHR and WKY dams and adult blood pressures of cross-fostered F1 pups. Dev Psychobiol 22, 55-67.
Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A. C., Cohen, S. N., and Numa, S. (1979). Nucleotide sequence of cloned cDNA for bovine corticotropin-beta-lipotropin precursor. Nature 278, 423-427.
Ohta, A., Takagi, H., Matsui, T., Hamai, Y., Iida, S., and Esumi, H. (1993). Localization of nitric oxide synthase-immunoreactive neurons in the solitary nucleus and ventrolateral medulla oblongata of the rat: their relation to catecholaminergic neurons. Neurosci Lett 158, 33-35.
Palmer, R. M., Ashton, D. S., and Moncada, S. (1988). Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333, 664-666.
Palmer, R. M., Ferrige, A. G., and Moncada, S. (1987). Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524-526.
Papapetropoulos, A., Rudic, R. D., and Sessa, W. C. (1999). Molecular control of nitric oxide synthases in the cardiovascular system. Cardiovasc Res 43, 509-520.
Pavia, J. M., Schioth, H. B., and Morris, M. J. (2003). Role of MC4 receptors in the depressor and bradycardic effects of alpha-MSH in the nucleus tractus solitarii of the rat. Neuroreport 14, 703-707.
Paxinos, G., and Watson, C. (1986). The rat brain in stereotaxic corrdinates, 2nd ed, Academic Press, New York.
Pritchard, L. E., Turnbull, A. V., and White, A. (2002). Pro-opiomelanocortin processing in the hypothalamus: impact on melanocortin signalling and obesity. J Endocrinol 172, 411-421.
Rees, D. D., Palmer, R. M., and Moncada, S. (1989). Role of endothelium-derived nitric oxide in the regulation of blood pressure. Proc Natl Acad Sci U S A 86, 3375-3378.
Reis, D. J. (1984). The brain and hypertension: reflections on 35 years of inquiry into the neurobiology of the circulation. Circulation 70, III31-45.
Reis, D. J., Granata, A. R., Perrone, M. H., and Talman, W. T. (1981). Evidence that glutamic acid is the neurotransmitter of baroreceptor afferent terminating in the nucleus tractus solitarius (NTS). J Auton Nerv Syst 3, 321-334.
Ruggiero, D. A., Mtui, E. P., Otake, K., and Anwar, M. (1996). Central and primary visceral afferents to nucleus tractus solitarii may generate nitric oxide as a membrane-permeant neuronal messenger. J Comp Neurol 364, 51-67.
Sakai, K., Hirooka, Y., Matsuo, I., Eshima, K., Shigematsu, H., Shimokawa, H., and Takeshita, A. (2000). Overexpression of eNOS in NTS causes hypotension and bradycardia in vivo. Hypertension 36, 1023-1028.
Spyer, K. M., Mifflin, S. W., and Withington-Wray, D. J. (1987). Organization of the autonomic nervous system: control and peripheral mechanisms. (New York, Alan R. Lisps).
Stuehr, D. J. (1999). Mammalian nitric oxide synthases. Biochim Biophys Acta 1411, 217-230.
Suzuki, I., Cone, R. D., Im, S., Nordlund, J., and Abdel-Malek, Z. A. (1996). Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis. Endocrinology 137, 1627-1633.
Talman, W. T., and Kelkar, P. (1993). Neural control of the heart. Central and peripheral. Neurol Clin 11, 239-256.
Titheradge, M. A. (1999). Nitric oxide in septic shock. Biochim Biophys Acta 1411, 437-455.
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.
Tseng, C. J., and Tung, C. S. (1995). Central cardiovascular regulation. In: Disorders of the Autonomic System, edited by Robertson, D. and Biaggioni, I., United Kindom, Harwood Academic Publisher.
Versteeg, D. H., Van Bergen, P., Adan, R. A., and De Wildt, D. J. (1998). Melanocortins and cardiovascular regulation. Eur J Pharmacol 360, 1-14.
Vincent, S. R., and Kimura, H. (1992). Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46, 755-784.
Wikberg, J. E. (1999). Melanocortin receptors: perspectives for novel drugs. Eur J Pharmacol 375, 295-310.
Wikberg, J. E., Muceniece, R., Mandrika, I., Prusis, P., Lindblom, J., Post, C., and Skottner, A. (2000). New aspects on the melanocortins and their receptors. Pharmacol Res 42, 393-420.
Zanzinger, J. (1999). Role of nitric oxide in the neural control of cardiovascular function. Cardiovasc Res 43, 639-649.