de Carvalho R P, de Faria M H, do Nascimento J L, Hokoç J N
Department of Neurobiology, Federal Fluminense University, Rio de Janeiro, Brazil.
J Neurochem. 1996 Sep;67(3):1063-71. doi: 10.1046/j.1471-4159.1996.67031063.x.
Nitric oxide plays an important role as an intercellular messenger in the CNS. In the present work we measured NADPH-diaphorase activity, which is considered to be a marker of cells producing nitric oxide, in homogenates of the developing chick retina. The enzyme activity can be detected beginning in 8-day-old embryonic retinas with no further quantitative variations throughout development. Arginine analogues inhibit approximately 65% of the activity in embryonic retinas and 50% in posthatched retinas. The enzyme is stimulated 50% by 2 mM calcium chloride in retinas from 8 to 14 embryonic days, but this effect decreases to 20% in 17-day embryonic retinas and practically disappears in posthatched animals. The stimulation by calcium is completely blocked by arginine analogues. The decrease in enzyme activity at posthatched retinas is not due to stimulation by endogenous calcium or the presence of insufficient amounts of calmodulin, because addition of EGTA or calmodulin, respectively, did not restore the stimulation to levels observed at embryonic stages. Inhibition of NADPH-diaphorase activity by NG-nitro-L-arginine or L-NG-(iminoethyl) ornithine is concentration dependent with IC50 values of approximately 1 mM at all stages studied. However, in the presence of calcium, the inhibition by both analogues is shifted to the left and is apparently biphasic at all developmental stages, including in posthatched animals, with IC50 values in the low micromolar range. NADPH-diaphorase was also detected by histochemistry in specific groups of cells in the early embryonic retina and in subsets of amacrine and ganglion cells, as well as in photoreceptors, in more developed retinas. The results indicate that different isoforms of nitric oxide synthase are present in the chick retina and that a calcium-dependent isoform is predominant in early periods of development.
一氧化氮作为一种细胞间信使在中枢神经系统中发挥着重要作用。在本研究中，我们测定了发育中的鸡视网膜匀浆中NADPH-黄递酶的活性，该酶活性被认为是产生一氧化氮的细胞的标志物。在8日龄胚胎视网膜中即可检测到该酶活性，且在整个发育过程中无进一步的定量变化。精氨酸类似物在胚胎视网膜中可抑制约65%的活性，在孵化后视网膜中可抑制50%的活性。在8至14日龄胚胎视网膜中，2 mM氯化钙可使该酶活性提高50%，但在17日龄胚胎视网膜中，这种作用降至20%，在孵化后动物中几乎消失。精氨酸类似物可完全阻断钙的刺激作用。孵化后视网膜中酶活性的降低并非由于内源性钙的刺激或钙调蛋白量不足，因为分别添加乙二醇双四乙酸（EGTA）或钙调蛋白并不能将刺激恢复到胚胎阶段观察到的水平。NG-硝基-L-精氨酸或L-NG-（亚氨乙基）鸟氨酸对NADPH-黄递酶活性的抑制作用呈浓度依赖性，在所研究的所有阶段，其半数抑制浓度（IC50）值约为1 mM。然而，在有钙存在的情况下，两种类似物的抑制作用均向左偏移，且在包括孵化后动物在内的所有发育阶段均表现为双相性，IC50值在低微摩尔范围内。通过组织化学方法还在早期胚胎视网膜的特定细胞群以及更发达视网膜中的无长突细胞和神经节细胞亚群以及光感受器中检测到了NADPH-黄递酶。结果表明，鸡视网膜中存在不同的一氧化氮合酶同工型，且钙依赖性同工型在发育早期占主导地位。
