Department of Biology and Biotechnology Charles Darwin, Sapienza University, 00161 Rome, Italy.
J Comp Neurol. 2012 Feb 1;520(2):364-83. doi: 10.1002/cne.22729.
Nitric oxide synthase (NOS) has been characterized in several opistobranchs and pulmonates but it was much less investigated in prosobranchs, which include more than 20,000 species and account for most of the gastropod diversity. Therefore, new data from this large group are needed for a better knowledge of the molecular evolution of NOS enzymes in molluscs. This study focused on NOS expressed in the nervous system of the prosobranch neogastropod Stramonita haemastoma. In this study we report compelling evidence on the expression of a constitutive Ca(2+) /CaM-dependent neuronal NOS in the central and peripheral nervous system. The prevailing neuronal localization of NADPHd activity was demonstrated by NADPHd histochemistry in both central and peripheral nervous system structures. L-arginine/citrulline assays suggested that Stramonita NOS is a constitutive enzyme which is both cytosolic and membrane-bound. Molecular cloning of the full-length Stramonita NOS (Sh-NOS) by reverse-transcription polymerase chain reaction (RT-PCR) followed by 5' and 3' RACE showed that Sh-NOS is a protein of 1,517 amino acids, containing a PDZ domain at the N-terminus and sharing similar regulatory domains to the mammalian neuronal NOS (nNOS). Regional expression of the Sh-NOS gene was evaluated by RT-PCR. This analysis showed different expression levels in the nerve ring, the osphradium, the cephalic tentacles, the buccal tissues, and the foot, whereas NOS expression was not found in the salivary glands and the gland of Leiblein. The present data provide a solid background for further studies addressing the specific functions of NO in neogastropods.
一氧化氮合酶（NOS）已在几种后鳃类和肺螺类中得到了描述，但在包括超过 20,000 个物种且占大多数腹足动物多样性的前鳃类中，研究较少。因此，需要来自这个大群体的新数据来更好地了解 NOS 酶在软体动物中的分子进化。本研究集中于前鳃类腹足纲 Neogastropoda Stramonita haemastoma 中表达的 NOS。在这项研究中，我们提供了令人信服的证据，证明了在中枢和周围神经系统中存在一种组成型 Ca(2+) /CaM 依赖性神经元 NOS。在中枢和周围神经系统结构中，通过 NADPHd 组织化学证明了 NADPHd 活性的主要神经元定位。L-精氨酸/瓜氨酸测定表明，Stramonita NOS 是一种组成型酶，既存在于细胞质中，也存在于膜结合中。通过逆转录聚合酶链反应（RT-PCR）和 5' 和 3' RACE 对全长 Stramonita NOS（Sh-NOS）的分子克隆表明，Sh-NOS 是一种含有 PDZ 结构域的 1,517 个氨基酸的蛋白质，其调控域与哺乳动物神经元 NOS（nNOS）相似。通过 RT-PCR 评估了 Sh-NOS 基因的区域表达。该分析显示，在神经环、触角、口腔组织和足部中，Sh-NOS 的表达水平不同，而在唾液腺和 Leiblein 腺中未发现 NOS 表达。目前的数据为进一步研究 NOS 在新腹足类中的特定功能提供了坚实的基础。
