pH and water regulation in the silk gland of Bombyx mori in relation to gel-sol transition of the liquid silk in the glandular lumen

丝腺是一个独特的和不可缺少的组织，生产丝蛋白（丝素和丝胶），使茧。腺分泌丝蛋白并暂时储存在腺腔内，直到幼虫开始吐丝，而蛋白质没有任何变性。分泌蛋白中的浓度达到20- 30%水平。这种高浓度的蛋白质溶液必须在一定的pH范围和固定的水含量下进行调节。我们已经研究了这样一种机制，以了解离子稳态和腺体中的钙离子调节。结果如下：（1）用V-ATPase特异性抗体进行免疫细胞化学研究。V-ATP酶在中部丝腺细胞（MSG）和前部丝腺细胞（ASG）中都有表达。V-ATP酶定位于细胞表面，表明该V-ATP酶是质膜型，而不是像溶酶体和分泌囊泡中的V-ATP酶那样的内膜型。(2)在另一种产茧昆虫蓖麻蚕（Samia cynthia ricini）中检测到类似的质膜V-ATPase，但在甘薯天蛾（Agrius convolvuli）的唇腺中未检测到，我们在其胞质中检测到内膜V-ATPase。因此，质膜V-ATPase的存在是鳞翅目昆虫丝腺的一个独特特征。(3)采用同源克隆的策略，部分克隆了家蚕丝腺水通道蛋白（aquaporin，AQP）。该蛋白在5龄幼虫取食期的ASG中高度表达。假定的水通道蛋白的表达似乎在旋转开始时停止。因此，AQP和V-ATPase与蚕蛹变态期丝腺的发育和产茧量密切相关。

The silk gland is a unique and indispensable tissue to produce the silk proteins (fibroin and sericin), making a cocoon. The gland secretes and temporarily stores the silk proteins within the glandular lumen without any denaturation of proteins till the larva starts to spin. The concentrations in the secreted protein reach up to 20-30 % levels. Such a high concentration of the protein solution has to be regulated under a certain pH ranges and a fixed water content. We have investigated such a mechanism to understand the ionic homeostasis and osmoregulation in the gland. Results are as follows :(1) Immunocytochemical studies were done with the specific anitibody against V-ATPase. The V-ATPase can be detected in the middle silk gland cell(MSG) as well as in the anterior silk gland (ASG) cell. V-ATPase was located at the cell surface, suggesting that this V-ATPase is a plasma membrane-type not an endomembrane-type such as the V-ATPase in lysosomes and secretory vesicles.(2) The similar plasma membrane V-ATPase detected in another cocoon-producing lepidopteran insect, Eri-silkworm (Samia cynthia ricini), but not in the labial gland from the sweet potato hornworm, Agrius convolvuli, where we have detected the endomembrane V-ATPase in the cytoplasm. Thus, the presence of plasma membrane V-ATPase is a unique feature in the silk gland of lepidoptera.(3) By the homology cloning strategy, we have patially cloned the putative water channel (aquaporin, AQP) in the silk gland of Bombyx mori. This putative AQP is highly expressed in the ASG at the feeding stage of the 5th instar. The expression of the putative AQP seems to be stopped at the onset of spinning. Thus, it is suggested that the AQP as well as V-ATPase is closely related to the silk gland development and the cocoon production during larval-pupal metamorphosis.