a-Adrenergic regulation of secretion of mouse saliva rich in nerve growth factor
a-Adrenergic regulation of secretion of mouse saliva rich in nerve growth factor
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α-肾上腺素能调节富含神经生长因子的小鼠唾液分泌
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发表时间：
2003
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通讯作者：
L. M. Partlow
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作者：
L. Wallace;L. M. Partlow
神经生长因子已定量生物测定和放射免疫扩散在小鼠唾液中引起的几个促分泌素。在肾上腺素和去甲肾上腺素诱导的唾液（3400和900微克/毫升，分别）的神经生长因子的生物测定浓度高于任何其他来源的报告。相反，异丙肾上腺素和毛果芸香碱诱导的唾液中的神经生长因子的浓度相对较低（分别为17和2 ;sg/ml）。唾液神经生长因子的比活性分别为41、36、2和0.6 ;肾上腺素、去甲肾上腺素、毛果芸香碱和异丙肾上腺素引起的分泌物中蛋白质的g/mg。肾上腺素或去甲肾上腺素给药后的流涎被α-肾上腺素能阻滞剂酚苄明完全抑制。这些结果表明，释放唾液丰富的神经生长因子主要是通过α-肾上腺素能受体调节。雄性小鼠的颌下腺是已知最丰富的神经生长因子（NGF）来源（1-3）。许多种蛇的毒液（4，5）和雌性小鼠的颌下腺（2，3）也是NGF的丰富来源。由于蛇毒是毒液腺的分泌产物，而毒液腺是哺乳动物唾液腺的系统发育同源物，早期的研究者也在小鼠唾液中寻找NGF。Levi-Montalcini和Cohen（6）报道，毛果芸香碱诱导的小鼠唾液中含有可检测水平的生物活性NGF，但估计浓度至少比唾液腺提取物低5000倍。这些研究仅检查了由副交感神经促分泌素诱导的唾液，尽管唾液分泌也由交感神经系统的活动引起。这种肾上腺素分泌物在蛋白质浓度和组成上与胆碱能唾液不同（7，8）。一些证据表明，肾上腺素能刺激后，神经生长因子可能从颌下腺分泌。这种生长因子与腺体分泌的许多其他蛋白质一起集中在曲颗粒小管中（9，10）：（i）肾素（或异肾素）（11，12），（ii）酯蛋白酶（13; 14）和（iii）表皮生长因子（EGF）（15，16）。在这些蛋白质中，酯蛋白酶和EGF均由肾上腺素能激动剂引起分泌到唾液中（14，16）。此外，Pasquini等人（17）已经证明，NGF、EGF和酯蛋白酶活性均与从小鼠颌下腺分离的相同细胞内颗粒相关。在本文和早期的摘要中，我们报道了肾上腺素或去甲肾上腺素引起的唾液分泌物中极高的NGF浓度，但异丙肾上腺素或毛果芸香碱没有引起。高浓度的神经生长因子在肾上腺素和去甲肾上腺素诱导的唾液已被量化的生物测定和径向免疫扩散。α-肾上腺素能受体阻滞剂酚苄明可抑制NGF的释放。因此，富含生物活性和免疫反应性的NGF的分泌物通过肾上腺素能激动剂对小鼠唾液腺内α-受体的作用而选择性地释放。材料与方法收集10 ~ 16周龄雄性小鼠的唾液。用60 mg/kg戊巴比妥麻醉动物。唾液分泌通常由腹腔注射促分泌素引起。每种促分泌剂的剂量范围为：毛果芸香碱，0.1-0.8 mg/kg;肾上腺素，2.0-6.6 mg/kg;去甲肾上腺素，0.7-2.5 mg/kg;异丙肾上腺素，0.25 mg/kg。在少数情况下，约十分之一的腹腔内剂量的促分泌素被注射到覆盖颌下腺的结缔组织鞘下。在所有实验中，在注射促分泌素后45分钟内，将合并的唾液分泌物收集在置于舌头和口底之间的微毛细管中。(The下颌下腺和舌下腺的导管通向舌下的口腔。收集后立即将唾液冷冻在-40°，直至进行测定。在促分泌素给药前30和60分钟，分别将抑制剂酚苄明和普萘洛尔以5 mg/kg注射到颈静脉中。NGF生物测定使用帕拉-
Nerve growth factor has been quantified by both bioassay and radial immunodiffusion in mouse saliva elicited by several secretagogues. The concentrations by bioassay of nerve growth factor in both epinephrineand norepinephrine-induced saliva (3400 and 900 gg/ml, respectively) are higher than reported in any other source. In contrast, the concentrations of nerve growth factor in isoproterenoland pilocarpine-induced saliva are relatively low (17 and 2 ;sg/ml, respectively). The specific activity of the salivary nerve growth factor was 41, 36, 2, and 0.6 ;&g/mg of protein in secretions elicited by epinephrine, norepinephrine, pilocarpine, and isoproterenol, respectively. Salivation after administration of either epinephrine or norepinephrine was completely inhibited by the a-adrenergic blocker, phenoxybenzamine. These results suggest that the release of saliva rich in nerve growth factor is primarily regulated through a-adrenergic receptors. The submaxillary gland of male mice is the richest known source of nerve growth factor (NGF) (1-3). Venom of numerous species of snakes (4, 5) and the submaxillary glands of female mice (2, 3) are also rich sources of NGF. Since snake venom is the secretory product of the venom gland, the phylogenetic homologue of the manmalian salivary gland, early investigators also looked for NGF in mouse saliva. Levi-Montalcini and Cohen (6) reported that pilocarpine-induced mouse saliva contained detectable levels of biologically active NGF, but estimated the concentration to be at least 5000 times lower than in salivary gland extracts. Those studies only examined saliva induced by a parasympathetic secretagogue, despite the fact that salivary secretions are also elicited by activity of the sympathetic nervous system. Such adrenergic secretions differ from cholinergic saliva in both protein concentration and composition (7, 8). Several lines of evidence suggest that NGF might be secreted from the submaxillary gland after adrenergic stimulation. This growth factor is concentrated in the convoluted granular tubules (9, 10) with a number of other proteins that are secreted by the gland: (i) renin (or isorenin) (11, 12), (ii) esteroproteases (13; 14), and (iii) epidermal growth factor (EGF) (15, 16). Of these proteins, both the esteroproteases and EGF are secreted in saliva elicited by adrenergic agonists (14, 16). In addition, Pasquini et al. (17) have demonstrated that NGF, EGF, and esteroprotease activity are all associated with the same intracellular granules isolated from the mouse submaxillary gland. In the present paper and in an earlier abstract (18), we report exceedingly high NGF concentrations in salivary secretions elicited by epinephrine or norepinephrine, but not by isoproterenol or pilocarpine. The high NGF concentrations in both epinephrineand norepinephrine-induced salivas have been quantified by both bioassay and radial immunodiffusion. NGF release was inhibited by the a-adrenergic blocker, phenoxybenzamine. Thus, a secretion rich in NGF that is both biologically active and immunologically reactive is selectively released by the action of adrenergic agonists on a-receptors within mouse salivary glands. MATERIALS AND METHODS Saliva was collected from 10to 16-week-old male mice. The animals were anesthetized with pentobarbital, 60 mg/kg. Salivation was usually induced by the intraperitoneal injection of secretagogue. The secretagogues and ranges of doses for each were: pilocarpine, 0.1-0.8 mg/kg; epinephrine, 2.0-6.6 mg/kg; norepinephrine, 0.7-2.5 mg/kg; and isoproterenol, 0.25 mg/kg. In a few cases about one-tenth the intraperitoneal dose of secretagogue was injected under the sheath of connective tissue covering the submaxillary gland. In all experiments, pooled salivary secretions were collected in a microcapillary tube placed between the tongue and the floor of the mouth over a 45-min period after injection of the secretagogue. (The ducts from the submaxillary and sublingual glands open into the buccal cavity under the tongue.) Immediately after collection, the saliva was frozen at -40° until the assays were performed. The inhibitors phenoxybenzamine and propranalol were injected at 5 mg/kg into the jugular vein 30 and 60 min, respectively, prior to administration of the secretagogue. The NGF bioassay was performed using cultures of para-