Nieto-Alamilla Gustavo, Escamilla-Sánchez Juan, López-Méndez María-Cristina, Molina-Hernández Anayansi, Guerrero-Hernández Agustín, Arias-Montaño José-Antonio
a Departamento de Fisiología, Biofísica y Neurociencias , Centro de Investigación y de Estudios Avanzados del IPN , Ciudad de México , México.
b Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN , Ciudad de México , México.
J Recept Signal Transduct Res. 2018 Apr;38(2):141-150. doi: 10.1080/10799893.2018.1448995. Epub 2018 Mar 20.
In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H receptor (hHR and hHR) on [S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca ions ([Ca]i) and depolarization-evoked [H]-dopamine release. Maximal specific binding (B) of [H]-N-methyl-histamine to cell membranes was 953 ± 204 and 555 ± 140 fmol/mg protein for SH-SY5Y-hHR and SH-SY5Y-hHR cells, respectively, with similar dissociation constants (K, 0.86 nM and 0.81 nM). The mRNA of the hHR isoform was 40.9 ± 7.9% of the hHR isoform. No differences in receptor affinity were found for the HR ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hHR cells ([S]-GTPγS binding, 158.1 ± 7.5% versus 136.5 ± 3.6% for SH-SY5Y-hHR cells; cAMP accumulation, -74.0 ± 4.9% versus -43.5 ± 5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [H]-dopamine release evoked by 100 mM K (-18.9 ± 3.0% and -20.5 ± 3.3%, for SH-SY5Y-hHR and SH-SY5Y-hHR cells), or the inhibition of depolarization-induced increase in [Ca]i (S2/S1 ratios: parental cells 0.967 ± 0.069, SH-SY5Y-hHR cells 0.639 ± 0.049, SH-SY5Y-hHR cells 0.737 ± 0.045). These results indicate that in SH-SY5Y cells, hHR and hHR isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.
在稳定转染的人神经母细胞瘤SH-SY5Y细胞中，我们比较了激活人组胺H受体（hHR和hHR）的445个和365个氨基酸的两种亚型对[S]-GTPγS结合、福斯可林诱导的cAMP形成、去极化诱导的细胞内钙离子浓度（[Ca]i）增加以及去极化诱发的[H]-多巴胺释放的影响。[H]-N-甲基组胺与细胞膜的最大特异性结合（B），SH-SY5Y-hHR细胞和SH-SY5Y-hHR细胞分别为953±204和555±140 fmol/mg蛋白，解离常数（K）相似（分别为0.86 nM和0.81 nM）。hHR亚型的mRNA是hHR亚型的40.9±7.9%。对于HR配体组胺（histamine）、依美哌啶（immepip）、（R）（-）-α-甲基组胺（RAMH）、A-331440、氯苯丙哌嗪（clobenpropit）和西普司他（ciproxifan），未发现受体亲和力存在差异。激动剂RAMH对[S]-GTPγS结合的刺激以及对福斯可林刺激的cAMP积累的抑制，在SH-SY5Y-hHR细胞中显著更大（[S]-GTPγS结合：SH-SY5Y-hHR细胞为158.1±7.5%，SH-SY5Y-hHR细胞为136.5±3.6%；cAMP积累：-74.0±4.9% vs -43.5±5.3%），对激动剂效力无显著影响。相反，RAMH抑制100 mM K诱发的[H]-多巴胺释放的效力和效果在SH-SY5Y-hHR细胞和SH-SY5Y-hHR细胞中无差异（分别为-18.9±3.0%和-20.5±3.3%），对去极化诱导的[Ca]i增加的抑制也无差异（S2/S1比值：亲代细胞为0.967±0.069，SH-SY5Y-hHR细胞为0.639±0.049，SH-SY5Y-hHR细胞为0.737±0.045）。这些结果表明，在SH-SY5Y细胞中，hHR和hHR亚型以不同方式调节受体激活触发的信号通路。
