KMUP-1對抗血清素誘發H9c2心肌細胞肥大的保護作用經由抑制L-型鈣離子通道

KMUP-1 Protects Against Serotonin -Induced Cardiac Hypertrophy via Inhibition of L-type Calcium Channels in H9c2 Cells

賴彥傑(Yan-Jie Lai)

指導教授 ： 吳炳男

高雄醫學大學/醫學院/藥理學科研究所/碩士(2010年)

https://doi.org/10.6832/KMU.2010.00037

摘要

血清素被認為在心臟疾病發展調控上扮演了一個重要的角色。KMUP-1是一個化學合成之嘌呤類衍生物，在先前的研究中發現，它不僅是鉀離子通道的活化劑，同時也是磷酸二酯酶 (PDE) 的抑制劑。然而KMUP-1對於心肌細胞 (H9c2) 上鈣離子通道的抑制作用尚未被直接證實過。
本實驗主要包括兩個研究目的，首先先探討KMUP-1在心肌細胞 (H9c2) 上，對於L型鈣離子通道是否具有抑制作用。其次再接著探討KMUP-1對於由血清素所引起的心肌細胞肥大及L型鈣離子通道活化的現象是否具有保護及抑制的作用。在H9c2細胞上投予10 μM血清素誘導4天，結果細胞表面積和對照組相比增加了55.5%， 而KMUP-1 (1 μM) 和ketanserin (0 .1 μM) 可以反轉這個作用。我們利用傳統全細胞膜電位箝制技術來研究在H9c2細胞上，通過L型鈣離子通道之鈣電流。於電位箝制狀態下，KMUP-1可以濃度相關性的抑制L型鈣離子通道電流。此外KMUP-1可以抑制由蛋白激酶C (PKC) 活化劑phorbol 12-myristate 13-acetate (PMA, 5 μM) 以及蛋白激酶A (PKA) 活化劑8-Br-cAMP (100 μM) 所增加的鈣電流。前處理給予蛋白激酶G (PKG) 抑制劑KT5823 (1 μM)，結果發現可以減少KMUP-1對於L型鈣離子通道電流的抑制作用。然而在正常H9c2細胞上，於浸浴溶液中加入10 μM血清素，發現可以增加鈣離子電流，而1 μM KMUP-1可以抑制由血清素活化的L型鈣離子通道電流。而在浸浴溶液中前處理蛋白激酶C抑制劑、蛋白激酶A抑制劑，chelerythrine (5 μM)、H-89 (5 μM)，發現可以減少血清素所增加的鈣電流。值得注意的是，在由血清素所誘導H9c2心肌細胞肥大的組別中發現，10 μM血清素誘導4天會使得L型鈣離子通道電流和對照組比較起來，增加了2.87倍。而在同時加入KMUP-1的組別發現，KMUP-1可以濃度相關性的抑制血清素所誘導L型鈣離子通道電流活化的現象。
最後，由以上實驗得知KMUP-1可以抑制心肌細胞 (H9c2) 上L型鈣離子通道電流，這個現象可能包含了參與蛋白激酶A、蛋白激酶C和蛋白激酶G的調控路徑。而血清素誘導心肌細胞肥大和活化L型鈣離子通道電流的作用，可能是藉由參與活化5HT2A受體、蛋白激酶A、蛋白激酶C和蛋白激酶G的調控路徑。而KMUP-1對於心肌細胞肥大的保護作用可能是藉由抑制L型鈣離子通道電流。而KMUP-1可能是經由調控蛋白激酶A、蛋白激酶C和蛋白激酶G的路徑，進一步抑制血清素所活化的L型鈣離子通道電流。

關鍵字

心肌細胞肥大 ； L型鈣離子通道 ； 血清素

並列摘要

Serotonin (5-hydroxytryptamine) is considered to play a role in the regulation of cardiac growth in pathological conditions. KMUP-1, a chemically synthetic xanthine-based derivative, has been demonstrated not only a K+ channels activator, but also a phosphodiesterases inhibitor. However, it has not been addressed in the inhibition of Ca2+ channels in H9c2 cardiomyocytes.
There are two aims of this study. First, it is to examine whether KMUP-1 has an inhibition of L-type Ca2+ channel (LTCC) in H9c2 cardiomyocytes. Second, it is to examine whether KMUP-1 can suppress the activation of LTCC and prevent the development of serotonin-induced hypertrophy in H9c2 cardiomyocytes. Rat heart-derived H9c2 cells were treated with serotonin (10 μM) for 4 days, the cell surface area was increased 55.5% and that was prevented by KMUP-1 (1 μM) and ketanserin (0.1 μM). Conventional whole cell patch-clamp technique was used to investigate Ca2+ currents through LTCC (ICa,L) in H9c2 cells. Under voltage-clamp conditions, KMUP-1 inhibited the ICa,L in a concentration-dependent manner. Additionally, KMUP-1 inhibited the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA, 5 μM) and 8-Br-cAMP (100 μM) enhanced ICa,L. Pretreatment with the PKG inhibitor KT5823 (1 μM) attenuated KMUP-1-inhibited ICa,L. However, in normal H9c2 cells, serotonin (10 μM) induced the ICa,L, and KMUP-1 (1 μM) also can inhibit serotonin-induced ICa,L. Pretreatment with the PKC and PKA inhibitors chelerythrine (5 μM), H-89 (5 μM) attenuated serotonin-induced ICa,L. Notably, the ICa,L was increased 2.87-fold in serotonin-induced hypertrophy in H9c2 cells. This increase in ICa,L was inhibited by KMUP-1 in a concentration-dependent manner.
In conclusion, KMUP-1 inhibits the ICa,L in H9c2 cells, which might in part involve the PKA, PKC and PKG pathways. Serotonin induces ICa,L activity that might in part involve the 5-HT2A receptor, PKA, PKC and PKG pathways. KMUP-1 also prevents serotonin-induced hypertrophy, which might attribute to its ICa,L inhibition. KMUP-1 inhibits serotonin-induced ICa,L activity that might in part involve the PKA, PKC and PKG pathways.

並列關鍵字

KMUP-1 ； cardiac hypertrophy ； serotonin ； L-type calcium channel ； H9c2

參考文獻

Akaike N, Kostyuk PG & Osipchuk YV (1989) Dihydropyridine-sensitive low-threshold calcium channels in isolated rat hypothalamic neurones. J Physiol 412, 181-195.

Alden KJ, Goldspink PH, Ruch SW, Buttrick PM & Garcia J (2002) Enhancement of L-type Ca2+ current from neonatal mouse ventricular myocytes by constitutively active PKC-betaII. Am J Physiol Cell Physiol 282, C768-774.

Aramburu J, Garcia-Cozar F, Raghavan A, Okamura H, Rao A & Hogan PG (1998) Selective inhibition of NFAT activation by a peptide spanning the calcineurin targeting site of NFAT. Mol Cell 1, 627-637.

Aramburu J, Yaffe MB, Lopez-Rodriguez C, Cantley LC, Hogan PG & Rao A (1999) Affinity-driven peptide selection of an NFAT inhibitor more selective than cyclosporin A. Science 285, 2129-2133.

Arikkath J & Campbell KP (2003) Auxiliary subunits: essential components of the voltage-gated calcium channel complex. Curr Opin Neurobiol 13, 298-307.