Berg K A, Clarke W P, Chen Y, Ebersole B J, McKay R D, Maayani S
Department of Anesthesiology, Mount Sinai School of Medicine, City University of New York, New York 10029.
Mol Pharmacol. 1994 May;45(5):826-36.
The effects of 5-hydroxytryptamine (5-HT)2A receptor activation on cAMP formation were studied in a cell line derived from embryonic rat cortex (A1A1). 5-HT (EC50 = 0.87 microM) amplified the amount of cAMP formed in response to 5'-N-ethylcarboxamidoadenosine (an adenosine A2 receptor agonist), cholera toxin, and forskolin after 15 min of coincubation in the presence of the phosphodiesterase inhibitor rolipram. This effect of 5-HT was blocked by 10 nM ketanserin as well as by 10 nM spiperone, indicating a response mediated by the 5-HT2A receptor subtype. Similarly, cAMP accumulation was enhanced by coincubation with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore A23187. After exposure to PMA for 24 hr (PKC-depleted cells), 5-HT and A23187 still enhanced cAMP formed in response to forskolin and 5'-N-ethylcarboxamidoadenosine, whereas the amplifying effects of PMA were abolished. Analysis by Western blots and PKC activity measurements revealed that, of three PKC isoforms detected in A1A1 cells (alpha, delta, and epsilon), only the calcium-independent isoform PKC-epsilon remained in membrane fractions after long term PMA treatment. In PKC-depleted cells, 5-HT-mediated amplification was greatly reduced after treatment with the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl)-ester or the calmodulin antagonists calmidazolium and N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride. In addition, 5-HT-mediated amplification of cAMP accumulation was reduced by the PKC inhibitor staurosporine in normal cells but was unaffected in PKC-depleted cells. In conclusion, these data suggest that 5-HT2A receptor activation can amplify cAMP formation in A1A1 cells by two distinct pathways coupled to the hydrolysis of inositol phosphates, i.e., PKC and calcium/calmodulin.
在源自胚胎大鼠皮质的细胞系（A1A1）中研究了5-羟色胺（5-HT）2A受体激活对环磷酸腺苷（cAMP）形成的影响。在磷酸二酯酶抑制剂咯利普兰存在的情况下共同孵育15分钟后，5-HT（半数有效浓度[EC50]=0.87微摩尔）增强了对5'-N-乙基羧基酰胺腺苷（一种腺苷A2受体激动剂）、霍乱毒素和福斯可林产生反应而形成的cAMP量。5-HT 的这种作用被10纳摩尔的酮色林以及10纳摩尔的螺哌隆阻断，表明这是由5-HT2A受体亚型介导的反应。同样，与蛋白激酶C（PKC）激活剂佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）和钙离子载体A23187共同孵育可增强cAMP的积累。在暴露于PMA 24小时（PKC耗竭细胞）后，5-HT和A23187仍然增强了对福斯可林和5'-N-乙基羧基酰胺腺苷产生反应而形成的cAMP，而PMA的放大作用则被消除。通过蛋白质印迹分析和PKC活性测量发现，在A1A1细胞中检测到的三种PKC同工型（α、δ和ε）中，长期PMA处理后只有不依赖钙的同工型PKC-ε保留在膜组分中。在PKC耗竭细胞中，用钙螯合剂1,2-双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸（乙酰氧甲基）酯或钙调蛋白拮抗剂氯咪达唑或盐酸N-（6-氨基己基）-5-氯-1-萘磺酰胺处理后，5-HT介导的放大作用大大降低。此外，PKC抑制剂星形孢菌素在正常细胞中降低了5-HT介导的cAMP积累放大作用，但在PKC耗竭细胞中则无影响。总之，这些数据表明，5-HT2A受体激活可通过与肌醇磷酸水解偶联的两条不同途径放大A1A1细胞中cAMP的形成，即PKC和钙/钙调蛋白途径。
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