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

丝腺是一种独特的、不可缺少的组织，可以产生丝蛋白（丝素和丝胶蛋白），从而形成茧。在幼虫开始纺丝之前，腺分泌并暂时将丝蛋白储存在腺腔内，而不会使蛋白质变性。分泌蛋白的浓度达到20- 30%的水平。如此高浓度的蛋白质溶液必须在一定的pH范围和固定的含水量下进行调节。我们研究了这一机制，以了解腺体中的离子稳态和渗透调节。结果如下：(1)用特异性抗体对V-ATPase进行了免疫细胞化学研究。v - atp酶可以在中间丝腺细胞（MSG）和前丝腺细胞（ASG）中检测到。V-ATPase位于细胞表面，提示该V-ATPase为质膜型，不像溶酶体和分泌囊泡中的V-ATPase那样为膜型。(2)在另一种产茧鳞翅目昆虫蚕（Samia cynthia ricini）中检测到类似的质膜v - atp酶，但在甘薯角虫（Agrius convolvuli）的唇腺细胞质中检测到膜v - atp酶。因此，质膜v - atp酶的存在是鳞翅目蛛丝腺的一个独特特征。(3)采用同源克隆策略，部分克隆了家蚕蚕丝腺中可能存在的水通道（aquaporin， AQP）。这一推测的AQP在5龄摄食期的ASG中高度表达。推测的AQP的表达似乎在旋转开始时停止。因此，AQP和V-ATPase与幼虫-蛹变态过程中蚕丝腺的发育和茧的产生密切相关。

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.