臺灣博碩士論文加值系統

中文摘要（Abstract in Chinese）

本論文主要探討(一)磷酸雙酯酶(phosphodiesterase, PDE)抑制劑，於非洲大蝸牛(Achatina fulica) 之RP4神經元自發性動作電位的影響。(二)進一步探討PDE抑制劑與d-amphetamine引發RP4神經元產生猝發現象之相關性。

(一)為探討PDE抑制劑對於中樞神經元的調控作用，使用特異性之PDE isoenzyme專一性抑制劑做測試，包括：caffeine (PDE non-selective), vinpocetine (PDE1 selective), EHNA (PDE2 selective), milrinone (PDE3 selective), rolipram (PDE4 selective), sildenafil (PDE5 selective)。

1.正常生理溶液灌流下，RP4神經元會產生規則的自發性動作電位(spontaneous action potential)。當投予投予PDE之抑制劑caffeine (30 mM), vinpocetine (300 μM), EHNA (1 mM), milrinone (300 μM), rolipram (300 μM)經灌流約30分鐘後，即可觀察到RP4神經元的動作電位由規律的自發性動作電位轉為猝發性(burst firing)動作電位。

2.若以細胞外灌流的方式，投與較高濃度的sildenafil (1 mM)，會使RP4神經元之自發性動作電位的頻率變慢，但無法產生猝發性動作電位。顯示：PDE抑制劑在RP4神經元的調控可能與cGMP無關。

3.投予rolipram (300 �嵱)引發RP4神經元產生的猝發現象後，以高鎂(30 mM)生理溶液進行灌流60分鐘後，此猝發現象仍然存在並未受到抑制。顯示: rolipram誘使RP4神經元產生的猝發現象，與在突觸前需要鈣離子(Ca2+-dependent)參與而釋放之神經傳遞物質無關；也與NMDA受體無關。由caffeine, vinpocetine, EHNA和 milrinone於RP4神經元所誘發之猝發現象也具相同反應。

4.若以KT5720 (10 �嵱)灌流RP4神經元60分鐘，再加入rolipram (300 �嵱)經60分鐘後，仍無猝發現象產生。顯示：KT5720 (10 �嵱)可以防止rolipram產生猝發性動作電位，而形成單一波峰動作電位，所以rolipram引起RP4神經元的猝發現象極有可能與cAMP-dependent protein kinase system的路徑有關。由caffeine, vinpocetine, EHNA和 milrinone於RP4神經元所誘發之猝發現象也具相同反應。

5.預先以rolipram (300 �嵱)引發RP4神經元產生猝發性動作電位後，接著投予phospholipase C抑制劑U73122 (10 �嵱)經2小時後，發現rolipram所引發RP4神經元產生的猝發現象並不改變。顯示: rolipram引起RP4神經元的猝發現象，並非經由活化phospholipase C而來的。由caffeine, vinpocetine, EHNA和 milrinone於RP4神經元所誘發之猝發現象也具相同反應。

6.以缺鈣生理溶液(Ca2+-free physiological solution)灌流RP4神經元20分鐘後，再加入rolipram (300 �嵱)投予灌流池內，經過20分鐘後，仍可發現猝發現象。顯示胞外鈣離子濃度與rolipram誘導所產生的猝發現象無關。由caffeine, vinpocetine, EHNA和 milrinone於RP4神經元所誘發之猝發現象也具相同反應。

7.利用膜電位箝制(voltage clamp)的實驗方式，測試rolipram對於總內向、外向離子電流和steady-state離子電流的影響，並作成電流與電位的關係圖(current-voltage relationship)，發現rolipram (100, 300 μM )對內向及外向電流的作用隨濃度愈高抑制的量愈多，具濃度依賴性；且rolipram (300 μM)引起RP4神經元產生猝發性動作電位後，其於steady-state外向離子電流的I-V圖上並不會引起negative slope resistance (NSR)。

(二)進一步探討PDE抑制劑與d-amphetamine引發RP4神經元產生猝發現象之相關性

1.投予較低濃度之d-amphetamine (67.5 μM)持續3小時後，不會引起RP4神經元產生猝發性動作電位，然而若投予d-amphetamine (270 μM)經30分鐘，即可誘發猝發現象產生。顯示d-amphetamine對膜電位的擺動現象(oscillation)及猝發性動作電位具有濃度依賴性。

2.預先給予caffeine (10 mM) 60分鐘，再接著投予較低濃度的d-amphetamine (67.5 �嵱)經60分鐘後，RP4神經元會產生猝發性動作電位。顯示: caffeine (10 mM)會加強低濃度的d-amphetamine (67.5 �嵱)引起動作電位的猝發現象。除此之外，vinpocetine (160 μM), EHNA (300 μM), milrinone (100 μM), rolipram (100 μM)，也可加強低濃度之d-amphetamine (67.5 μM)於RP4神經元產生猝發性動作電位。顯示抑制PDE的活性可促進d-amphetamine引發猝發現象。

3.預先給予高濃度之sildenafil (viagra, 1 mM)灌流60分鐘，再接著投予較低濃度的d-amphetamine (67.5 �嵱)經2小時後，也無法引起RP4神經元產生猝發性動作電位。顯示高濃度之sildenafil (1 mM)並不會促進低濃度的d-amphetamine (67.5 �嵱)引起動作電位的猝發。所以推測PDE type 5對d-amphetamine所引起猝發現象的調控較無相關。

本論文實驗結果顯示：在非洲大蝸牛的RP4神經元上，細胞外投予PDE抑制劑caffeine, vinpocetine, EHNA, milrinone, rolipram會產生猝發性動作電位，此一猝發現象不受胞外鈣離子濃度影響，也非經由興奮NMDA受體或增加神經傳導物質之釋放而產生，而與活化cAMP-dependent protein kinase system的路徑有關，且並不需phospholipase C的活化。因sildenafil無法引起RP4神經元產生猝發性動作電位，所以PDE抑制劑在RP4神經元的調控可能也與cGMP無關。由於rolipram對離子電流的抑制作用具有濃度依賴性，顯示PDE抑制劑經訊息傳遞作用可能調控離子電流的表現。而預先給予低濃度PDE抑制劑caffeine, vinpocetine, EHNA, milrinone, rolipram可促進d-amphetamine產生猝發性動作電位，可知PDE的活性興d-amphetamine之猝發現象相關。

The roles of phosphodiesterase (PDE) on the spontaneous action potentials and the effects of phosphodiesterase (PDE) inhibitors on bursts of potential elicited by d-amphetamine were studied on RP4 neuron of snail, Achatina fulica Ferussac in vitro. PDE inhibitors used include: vinpocetine (PDE1 selective), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, PDE2 selective), milrinone (PDE3 selective), rolipram (PDE4 selective), sildenafil (viagra) (PDE5 selective) and the non-selective inhibitor, caffeine. Vinpocetine, EHNA, milrinone, rolipram and caffeine but not sildenafil (viagra), did elicited bursts of potentials on the RP4 neuron. Calcium free solution or high magnesium solution or U73122 did not block the bursts of potential elicited by rolipram. However, the bursts of potential elicited by rolipram were blocked by KT5720. Similar effects were also found in vinpocetine, EHNA, milrinone and caffeine elicited bursts of potential. These results suggest that vinpocetine, EHNA, milrinone, rolipram or caffeine elicited bursts of potential through cAMP signalling pathways in central neuron of snail.
The ionic currents of the RP4 neuron were measured under two electrode voltage clamping. Rolipram significantly decreased the amplitude of total inward currents and outward currents in a concentration-dependent manner, but did not elicit a negative slope resistance (NSR) in current-voltage relationships of steady-state outward currents. These results suggest that the effect was in associate with the phosphodiesterase activity in the neuron. It is concluded that the phosphodiesterase activity plays an important role on the modulation of potentials in the RP4 neuron.
Oscillation of membrane potential and bursts of potential were elicited by d-amphetamine, vinpocetine, EHNA, milrinone, rolipram and caffeine in a concentration-dependent manner, while sildenafil (viagra) did not elicit bursts of potential on the RP4 neurons. Caffeine (10 mM), vinpocetine (160 μM), EHNA (300 μM), milrinone (100 μM), rolipram (100 μM) and forskolin (100 μM) potentiate the bursts of potential elicited by d-amphetamine. Sildenafil (Viagra, 1 mM) did not affect the bursts of potentials elicited by d-amphetamine. It is concluded that the bursts of potential elicited by d-amphetamine was associated with the phosphodiesterase activity in the neuron.

目錄 (Contents) :
頁數
一、縮寫表(Abbreviation)ii

二、中文摘要(Abstract in Chinese)1

三、英文摘要(Abstract in English)5

四、緒論(Introduction)7

五、實驗方法與材料(Materials and Methods)11

六、實驗結果(Results)

第1章 磷酸雙酯酶(PDE)抑制劑對RP4神經元膜電位的影響14
第2章 磷酸雙酯酶(PDE)抑制劑對d-amphetamine引發動作電
位猝發影響27

七、討論與結論(Discussion and Conclusion)34

八、參考文獻(References)44

九、圖表(Figures and Tables)50

十、附表(Appendix)86

Akaike, N., Furukawa, K., Kogure, K., 1993. Rolipram enhances the development of voltage-dependent Ca2+ current and serotonin-induced current in rat pheochromocytoma cells. Brain Res. 620, 58-63.
Altrup, U., Hader, M., Storz, U., 2003. Endogenous pacemaker potentials develop into paroxysmal depolarization shifts (PDSs) with application of an epileptogenic drug. Brain Res. 975, 73-84.
Arvanov, V.L., Chen, R.C., Chen, Y.H., Liou, H.H., Chan, Y.C., Arvanova, V.A., Tsai, M.C., 1994. Modulation of pentylenetetrazol induced bursting activity by electrogenic Na pump in Achatina fulica neurons. Asia Pacific J. Pharmacol. 9, 37-42.
Bardou, M., Goirand, F., Bernard, A., Guerard, P., Gatinet, M., Devillier, P., Dumas, J.P., Morcillo, E.J., Rochette, L., Dumas, M., 2002. Relaxant effects of selective phosphodiesterase inhibitors on U46619 precontracted human intralobar pulmonary arteries and role of potassium channels. J. Cardiovasc. Pharmacol. 40, 153-161.
Beavo, J.A., 1995. Cyclic nucleotide phosphodiesterases: Functional implications of multiple isoforms. Physiol. Rev. 75, 725–748.
Bonoczk, P., Gulyas, B., Adam-Vizi, V., Nemes, A., Karpati, E., Kiss, B., Kapas, M., Szantay, C., Koncz, I., Zelles, T., Vas, A., 2000. Role of sodium channel inhibition in neuroprotection: effect of vinpocetine. Brain Res. Bull. 53, 245-254.
Chen, Y.H., Tsai, M.C., 1996. Bursting firing of action potential in central snail neuron elicited by d-amphetamine: role of calcium ions. Comp. Biochem. Physiol. 115A, 195 – 205.
Chen, Y.H., Tsai, M.C., 1997. Bursting firing of action potentials in central snail neuron elicited by d-amphetamine: role of cytoplasmic second messengers. Neurosci. Res. 27, 295-304.
Chen, Y.H., Tsai, M.C., 2000. Action potential bursts in central snail neurons elicited by d-amphetamine: role of ionic currents. Neuroscience 96, 237-248.
Essayan, D.M., 2001. Cyclic nucleotide phosphodiesterases. J. Allergy Clin. Immunol. 108, 671-680.
Gainer, H., 1972. Electrophysiological behavior of an endogenously active neurosecretory cell. Brain Res. 39, 403-418.
Geelen, P., Drolet, B., Rail, J., Berube, J., Daleau, P., Rousseau, G., Cardinal, R., O''Hara, G.E., Turgeon, J., 2000. Sildenafil (Viagra) prolongs cardiac repolarization by blocking the rapid component of the delayed rectifier potassium current. Circulation 102, 275-277.
Goding, J.W., Grobben, B., Slegers, H., 2003. Physiological and pathophysiological functions of the ecto-nucleotide pyrophosphatase/phosphodiesterase family. Biochim. Biophys. Acta. 1638, 1-19.
Gorman, A.L.F., Hermann, A., 1982. Quantitative differences in the currents of bursting and beating molluscan pacemaker neurons. J. Physiol. (Lond) 333, 681-699.
Gorman, A.L.F., Thomas, A., Thomas, M.V., 1981. Intracellular calcium and control of neuronal pacemaker activity. Fed. Proc. 40, 2233-2239.
Greengard, P., 1978. Phosphorylated proteins as physiological effectors. Science 199, 146-152.
Higashima, M., Ohno K., Koshino, Y., 2002. Cyclic AMP-mediated modulation of epileptiform afterdischarge generation in rat hippocampal slices. Brain Res. 949, 157-161.
Hille, B., 1992. Ionic Channels of Excitable Membranes, 2nd edn, Sinauer Press, Sunderland, MA.
Jin, W., Sugaya, A., Tsuda, T., Ohguchi, H., Sugaya, E., 2000. Relationship between large conductance calcium-activated potassium channel and bursting activity. Brain Res. 860, 21-28.
Kajimoto, K., Hagiwara, N., Kasanuki, H., Hosoda, S., 1997. Contribution of phosphodiesterase isozymes to the regulation of the L-type calcium current in human cardiac myocytes. Br. J. Pharmacol. 121, 1549-1556.
Kakkar, R., Raju, R.V., Sharma, R.K., 1999. Calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1). Cell Mol. Life Sci. 55, 1164-1186.
Kerkut, G.A., Lambert, J.D.C., Gayton, R.J., Loker, J.E., Walker, R.J., 1975. Mapping of nerve cells in the subesophageal ganglia of Helix aspersa. Comp. Biochem. Physiol. 50A, 1-25.
Lancaster, B., Nicoll, R.A., 1987. Properties of two calcium-activated hyperpolarizations in rat hippocampal neurons. J. Physiol. (Lond) 389, 187-203.
Leibowitz, M.D., Sutro, J.B., Hille, B., 1986. Voltage-dependant gating of veratridine-modified Na channels. J. Gen. Physiol. 87, 25-46.
Liu, M.C., 2003. Effects of a New Isoquinolinone Derivative on the Action Potentials of Snail Central Neurons. Master’s Thesis, Department of Pharmacology, National Taiwan University.
Maurice, D.H., Palmer, D., Tilley, D.G., Dunkerley, H.A., Netherton, S.J., Raymond, D.R., Elbatarny, H.S., Jimmo, S.L., 2003. Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system. Mol. Pharmacol. 64, 533-546.
Meech, R.W., 1974. Prolonged action potentials in Aplysia neurons injected with EGTA. Comp. Biochem. Physiol. 48A, 397-402.
Mery, P.F., Pavoine, C., Pecker, F., Fischmeister, R., 1995. Erythro-9-(2-hydroxy-3-nonyl)adenine inhibits cyclic GMP-stimulated phosphodiesterase in isolated cardiac myocytes. Mol. Pharmacol. 48, 121-130.
Morner, S.E., Arlock, P., 1994. Mechanical and electrophysiological effects of milrinone on the force-frequency relationship in mammalian myocardium. Acta Physiol. Scand. 150, 125-132.
Onozuka, M., Kubo, K.A., Ozono, S., 1991a. The molecular mechanism underlying pentylenetetrazole-induced bursting activity in Euhadra neurons: involvement of protein phosphorylation. Comp. Biochem. Physiol. 100C, 423-432.
Onozuka, M., Imai, S., Furuichi, H., Ozono, S., 1991b. A specific 70K protein found in epileptic rat cortex: induction of bursting activity and negative resistance by its intracellular application in Euhadra neurons. J. Neurobiol. 22, 287 - 297.
Penner, R., Mathews, G., Neher, E., 1988. Regulation of calcium influx by second messengers in rat mast cells. Nature 334, 499-504.
Podzuweit, T., Nennstiel, P., Muller, A., 1995. Isozyme selective inhibition of cGMP-stimulated cyclic nucleotide phosphodiesterases by erythro-9-(2-hydroxy-3-nonyl) adenine. Cell Signal 7, 733-738.
Reid, I.A., 1999. Role of phosphodiesterase isoenzymes in the control of renin secretion: effects of selective enzyme inhibitors. Curr. Pharm. Des. 5, 725-735.
Reinikainen, K.J., Pitkanen, A., Riekkinen, P.J., 1989. 2'',3''-cyclic nucleotide-3''-phosphodiesterase activity as an index of myelin in the post-mortem brains of patients with Alzheimer''s disease. Neurosci. Lett. 106, 229-232.
Reynolds, I.J., 1990. Modulation of NMDA receptor responsiveness by neurotransmitters, drug and chemical modification. Life Sci. 47, 1785-1972.
Rosen, R.C., McKenna, K.E., 2002. PDE-5 inhibition and sexual response: pharmacological mechanisms and clinical outcomes. Annu. Rev. Sex Res. 13, 36-88.
Scholz, K.P., Byrne, J.H., 1988. Intracellular injection of cAMP induced a long-term reduction of neuronal K+ current. Science 240, 1664-1666.
Schrofner, S., Zsombok, A., Hermann, A., Kerschbaum, H.H., 2004. Nitric oxide decreases a calcium-activated potassium current via activation of phosphodiesterase 2 in Helix U-cells. Brain Res. 999, 98-105.
Schwartzkroin, P.A., 1994. Cellular electrophysiology of human epilepsy. Epilepsy Res. 17, 185-192.
Solntseva, E.I., Bukanova Yu, V., 1999. Cyclic GTP imitates the potentiating effect of the nootrope vinpocetine on the high-threshold A-current in mollusk neurons. Neurosci. Behav. Physiol. 29, 671-675.
Staveren, W.C., Markerink, M., Steinbusch, H.W., 2001. The effects of phosphodiesterase inhibition on cyclic GMP and cyclic AMP accumulation in the hippocampus of the rat. Brain Res. 888, 275-286.
Stork, P.J., Schmitt, J.M., 2002. Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol. 12, 258-66.
Sugaya, E., Onozuka, M., 1978a. Intracellular calcium: its movement during pentylenetetrazol-induced bursting activity. Science 200, 797-799.
Sugaya, E., Onozuka, M., 1978b. Intracellular calcium: its release from granules during bursting activity in snail neurons. Science 202, 1195-1197.
Sugaya, A., Sugaya, E., Tsujtani, M., 1973. Pentylenetetrazol-induced intracellular potential changes of the neuron of the Japanese land snail Enhadra peliomphala. Jap. J. Physiol. 23, 261-274.
Suvarna, N.U., O''Donnell J.M., 2002. Hydrolysis of N-methyl-D-aspartate receptor-stimulated cAMP and cGMP by PDE4 and PDE2 phosphodiesterases in primary neuronal cultures of rat cerebral cortex and hippocampus. J. Pharmacol. Exp. Ther. 302, 249-256.
Takeuchi, H., Araki, Y., Emaduddin, M., Zhang, W., Han, X.Y., Salunga, T.L., Wong, S.M., 1996. Identifiable Achatina giant neurones: their localizations in ganglia, axonal pathways and pharmacological features. Gen. Pharmacol. 27, 3-32.
Taylor, C.W., Marshall, I.C., 1992. Calcium and inositol 1,4,5-trisphosphate receptors: a complex relationship. Trends Biochem. Sci. 17, 403-407.
Thomas, M.V., Gorman, A.L.F, 1977. Internal calcium changes in a bursting pacemaker neuron measured with arsenazo Ⅲ. Science 196, 531-533.
Tsai, M.C., Chen, M.L., 1989. A new method for screening anticonvulsants: Eeffects of anticonvulsants on pentylenetetrazol-induced neuronal activity of the giant African snail, Achatina fulica Ferussac. Asia Pacific J. Pharmacol. 4, 203-207.
Tsai, M.C., Chen, Y.H., 1995. Bursting firing of action potential in central snail neuron elicited by d-amphetamine: role of electrogenic sodium pump. Comp. Physiol. Pharmacol. 111C, 131-141.
Yu, X., Byrne, J.H., Baxter, D.A., 2004. Modeling interactions between electrical activity and second-messenger cascades in Aplysia neuron R15. J. Neurophysiol. 91, 2297-2311.
Wang, S.J., Sihra, T.S., 2003. Opposing facilitatory and inhibitory modulation of glutamate release elicited by cAMP production in cerebrocortical nerve terminals (synaptosomes). Neuropharmacology 44, 686-697.
Watanabe, K., Funase, K., 1991. Cyclic AMP elicits biphasic current whose activation is mediated through protein phosphorylation in snail neurons. Neurosci. Res. 10, 64-70.
Wolfart, J., Roeper, J., 2002. Selective coupling of T-type calcium channels to SK potassium channels prevents intrinsic bursting in dopaminergic midbrain neurons. J. Neurosci. 22, 3404-3413.
Wu, C.L., 2002. Effects of ATP-sensitive K+ channel blockers on the action potentials of central neurons of Achatina fulica Ferussac. Master’s Thesis, Department of Pharmacology, National Taiwan University.
Zagnoni, P.G., Albano, C., 2002. Psychostimulants and epilepsy. Epilepsia 43, 28-31.