Marsigliante S, Muscella A, Greco S, Elia M G, Vilella S, Storelli C
Dipartimento di Biologia, Laboratorio di Fisiologia, Università di Lecce, Via Provinciale per Monteroni, 73100 Lecce, Italy.
J Endocrinol. 2001 Feb;168(2):339-46. doi: 10.1677/joe.0.1680339.
In the eel, angiotensin II (Ang II) has a role at the level of both gill chloride and kidney tubular cells, regulating sodium balance and therefore osmoregulation. The present study extends these findings to another important osmoregulatory organ - the intestine. Enterocytes were obtained from sea-water (SW)-acclimated eels to investigate the role of Ang II on the intestinal Na+/K+ATPase activity, because in SW-acclimated animals the intestine represents an important site of water and NaCl transport from the mucosal to the serosal side. This paper demonstrates that isolated enterocytes stimulated with increasing Ang II concentrations (0.01-100 nM) showed a dose-dependent inhibition of the Na+/K+ATPase activity. The threshold decrease was at 0.01 nM Ang II; it reached a maximum at 10 nM (81.5% inhibition) and did not decrease further with the use of higher hormone doses. These hormonal effects were blocked by a specific competitive antagonist of the AT1 receptor subtype, DuP-753 (100% inhibition at 10 microM), indicating that these effects are mediated by an AT1-like receptor. Isolated enterocytes stimulated with 10 nM Ang II showed a transient increase in intracellular calcium ([Ca2+]i), followed by a lower sustained phase. Removal of extracellular Ca2+ did not reduce the initial transient response and completely abolished the plateau phase. The inhibition of the Na+/K+ATPase activity was dependent on protein kinase C (PKC) activation since PKC antagonists (calphostin C and staurosporine) abolished the inhibitory effect of Ang II, and the PKC activator phorbol 12-myristate 13-acetate reduced transporter activity. Western blot analysis with antibodies to PKC alpha, beta I, beta II, gamma, delta, epsilon, iota, eta and zeta isoforms showed that eel enterocytes expressed the conventional isoforms (alpha and beta I), the novel isoforms (delta and eta) and the atypical isoforms (zeta and iota). Ang II stimulated the translocation from the cytosol to the plasma membrane of PKC alpha, PKC delta and PKC eta isoforms. In conclusion, our results suggest that Ang II modulates intestinal Na+/K+ATPase in SW-acclimated eels via calcium mobilization and PKC activation.
在鳗鱼中，血管紧张素II（Ang II）在鳃氯化物细胞和肾小管细胞水平上发挥作用，调节钠平衡，进而调节渗透压。本研究将这些发现扩展到另一个重要的渗透压调节器官——肠道。从适应海水（SW）的鳗鱼中获取肠上皮细胞，以研究Ang II对肠道Na+/K+ATP酶活性的作用，因为在适应SW的动物中，肠道是水和NaCl从黏膜侧转运至浆膜侧的重要部位。本文表明，用递增浓度（0.01 - 100 nM）的Ang II刺激分离的肠上皮细胞，Na+/K+ATP酶活性呈剂量依赖性抑制。阈值降低发生在0.01 nM Ang II时；在10 nM时达到最大抑制（81.5%抑制），使用更高激素剂量时不再进一步降低。这些激素效应被AT1受体亚型的特异性竞争性拮抗剂DuP - 753阻断（在10 microM时100%抑制），表明这些效应是由类似AT1的受体介导的。用10 nM Ang II刺激分离的肠上皮细胞，细胞内钙（[Ca2+]i）呈现短暂升高，随后是较低的持续阶段。去除细胞外钙不会降低初始短暂反应，但会完全消除平台期。Na+/K+ATP酶活性的抑制依赖于蛋白激酶C（PKC）激活，因为PKC拮抗剂（钙泊三醇C和星形孢菌素）消除了Ang II的抑制作用，且PKC激活剂佛波酯12 - 肉豆蔻酸13 - 乙酸酯降低了转运体活性。用针对PKCα、βI、βII、γ、δ、ε、ι、η和ζ同工型的抗体进行蛋白质印迹分析表明，鳗鱼肠上皮细胞表达传统同工型（α和βI）、新型同工型（δ和η）和非典型同工型（ζ和ι）。Ang II刺激PKCα、PKCδ和PKCη同工型从细胞质向质膜的转位。总之，我们的结果表明，Ang II通过钙动员和PKC激活来调节适应SW的鳗鱼肠道中的Na+/K+ATP酶。
