Allaerts W, Koopman W J, Verlaan B P, Buzzi M, Steerenberg P A
Department of Cellular Animal Physiology, Nijmegen Institute for Neurosciences, University of Nijmegen, Toernooiveld 1, Nijmegen, 6525 ED, The Netherlands.
Nitric Oxide. 2000 Feb;4(1):15-28. doi: 10.1006/niox.1999.0266.
Previous studies have focused on the immunohistochemical detection of a nitric oxide (NO)-cyclic 3',5'-monophosphate (cGMP) pathway in the brain and pituitary of the aquatic toad Xenopus laevis. We here investigate the endogenous production and possible involvement of NO signaling in the regulation of melanotrope cell activity in the pituitary pars intermedia of this amphibian. Using immunohistochemical staining of cultured cells with a polyclonal antiserum against inducible NO synthase (iNOS), immunoreactivity was observed both in melanotropes and in stellate-shaped cells. Part of these stellate-shaped cells is characterized as folliculo-stellate cells by their capacity of beta-Ala-Lys-N(epsilon)-AMCA uptake. Using chemiluminescence detection we demonstrate the presence of NO and reaction products like nitrite (NO(-)(2)) or peroxynitrite (ONOO(-)) in the incubation medium of cultured melanotropes. Bacterial lipopolysaccharide (LPS) stimulates the generation of NO and reaction products, the effect of which was blocked by S-methyl-l-thiocitrulline hydrochloride, a potent general NOS inhibitor. With [(3)H]lysine incorporation and a superfusion technique, it is shown that peptide release from melanotropes is stimulated by administration of superoxide dismutase (SOD), which was added to the superfusion medium to prevent scavenging of NO by superoxide anions. Pretreating the cells with the general NOS inhibitor l-nitroarginine methyl ester for 48 h attenuated the SOD-induced stimulation, but did not affect the stimulation by sodium nitroprusside (SNP) or 3-morpholinylsydnoneimine chloride (SIN-1), whereas hemoglobin blocked the combined effect of SOD plus NO donors. The soluble guanylate cyclase inhibitor 1H-[1,2, 4]oxadiazolo[4,3a]-quinoxaline-1-one did not inhibit but even significantly potentiated the effect of NO donors on peptide release without affecting the SOD-induced stimulation of peptide release. In addition to the previously described neuronal NOS (nNOS) immunoreactivity in nerve fibers in the pars intermedia of Xenopus, the present data reveal iNOS and nNOS as potential sources of endogenous NO production in cultured cells of the pars intermedia. Our study shows that also in nonmammalian vertebrates endogenous NO production may be physiologically relevant under conditions where protection against oxidative damage is needed. The endocrine cells of the pars intermedia themselves, as well as the folliculo-stellate cells, under such conditions may dispose of a protective mechanism against oxidative stress. The sensitivity of the endogenous NO production to LPS suggests that NO may also play a role during systemic inflammation.
以往的研究主要集中在对水生蟾蜍非洲爪蟾大脑和垂体中一氧化氮（NO）-环磷酸鸟苷（cGMP）途径进行免疫组织化学检测。我们在此研究了该两栖动物垂体中间叶中黑素细胞活性调节过程中NO信号的内源性产生及其可能的作用。使用针对诱导型一氧化氮合酶（iNOS）的多克隆抗血清对培养细胞进行免疫组织化学染色，在黑素细胞和星状细胞中均观察到免疫反应性。这些星状细胞中的一部分通过摄取β-丙氨酰-赖氨酸-N（ε）-AMCA的能力被鉴定为滤泡-星状细胞。使用化学发光检测法，我们证明了在培养的黑素细胞的孵育培养基中存在NO以及亚硝酸盐（NO₂⁻）或过氧亚硝酸盐（ONOO⁻）等反应产物。细菌脂多糖（LPS）刺激NO和反应产物的生成，而强效的通用NOS抑制剂盐酸S-甲基-L-硫代瓜氨酸可阻断其作用。通过[³H]赖氨酸掺入和灌流技术表明，向灌流培养基中添加超氧化物歧化酶（SOD）以防止超氧阴离子清除NO，可刺激黑素细胞释放肽。用通用NOS抑制剂L-硝基精氨酸甲酯预处理细胞48小时可减弱SOD诱导的刺激，但不影响硝普钠（SNP）或3-吗啉代亚磺酰亚胺氯化物（SIN-1）的刺激，而血红蛋白可阻断SOD加NO供体的联合作用。可溶性鸟苷酸环化酶抑制剂1H-[1,2,4]恶二唑并[4,3-a]喹喔啉-1-酮不抑制但甚至显著增强NO供体对肽释放的作用，而不影响SOD诱导的肽释放刺激。除了先前描述的非洲爪蟾中间叶神经纤维中的神经元型一氧化氮合酶（nNOS）免疫反应性外，本研究数据还揭示iNOS和nNOS是中间叶培养细胞中内源性NO产生的潜在来源。我们的研究表明，在需要防止氧化损伤的情况下，非哺乳动物脊椎动物内源性NO的产生在生理上也可能具有相关性。在这种情况下，中间叶的内分泌细胞本身以及滤泡-星状细胞可能具有一种针对氧化应激的保护机制。内源性NO产生对LPS的敏感性表明NO在全身炎症过程中也可能起作用。
