Role of the Calcium Sensing Receptor in Meal Stimulated Gastrin Secretion

对摄入的营养物质的分子识别导致胃肠激素的分泌，调节消化和肠道动力，是吸收这些营养物质所必需的。氨基酸、短链脂肪酸、碳水化合物和管腔内pH的变化是已知的胃泌素、缩胆囊素和促胰液素的有力刺激因素，这些激素是调节消化、分泌和运动的一些最重要的胃肠激素。负责这些营养素的化学感觉的细胞被推定分布在胃肠道内的粘膜层上，可能是肠道神经系统内的神经元。这些化学感觉细胞固有的分散性以及缺乏任何已知的标记来鉴定它们，为解决营养识别的分子基础创造了一个困难的问题。研究表明，作用于胃G细胞上钙感受器的钙能刺激胃泌素的分泌。除了二价阳离子外，CaSR还被证明是氨基氨基酸和pH的多模式传感器。因此，我们推测胃窦G细胞上表达的胃钙感受器(CaSR)是体内调节胃泌素释放的生理相关的感受器，介导蛋白质、氨基酸(尤其是芳香族氨基酸)和pH的释放。 本研究利用了Martin R.Pollak(JCI 111：1021-1028,2003)提供的CaSR和甲状旁腺激素(PTH)基因缺失小鼠。研究了CaSR基因纯合子缺失(KO)(CaSR-/-/PTH-/-)、杂合子(HET+/-/PTH-/-)和野生型(WT)(CaSR+/+/PTH-/-)小鼠血浆胃泌素对蛋白胨、苯丙氨酸(Phe)和pH的反应。小鼠禁食过夜，自由喂养，或以蛋白胨(8%)、苯丙氨酸(100 MM)、庚二烯(150 mM，pH 7.0)或氨甲酰胆碱(5 Mg)或蛙皮素(50 ug/kg)灌胃，用放射免疫法测定每种药物的胃泌素峰值反应。测定胃泌素-17(1 mg/kg)、组胺(10 mg/kg)或卡巴胆碱(5 mg/kg)对胃酸分泌的影响。用兔抗胃泌素对胃冰冻切片进行苏木精-伊红染色和免疫组织化学染色。 我们发现CaSR缺陷小鼠不会分泌胃泌素，对钙、蛋白胨、L-苯丙氨酸和升高的pH的腔内刺激有反应。然而，CaSR缺陷小鼠对注射蛙皮素和氨甲酚的反应与WT小鼠相似。杂合子和WT仔鼠对自由和灌胃喂养的胃泌素反应与WT C57/B6对照小鼠相似。高Ca~(++)饮食纠正CaSR缺乏小鼠血浆Ca~(++)不能恢复其胃泌素反应。CaSR缺陷小鼠胃窦G细胞数量减少，胃泌素含量减少。尽管它们对外源胃泌素和组胺表现出正常的胃酸分泌反应，但它们的静息胃pH也升高，基础酸分泌减少。 从这些研究中我们了解到，胃CASR对于大鼠胃腔内营养物质的生理感觉是必需的，如大鼠的食物、蛋白胨、Phe和导致胃窦G细胞胃泌素释放的pH升高。这些刺激物的感觉是否由胃窦G细胞上的CaSRs直接介导，还有待利用分离的G细胞进行进一步的研究。此外，我们还发现CaSR在决定胃窦G细胞数量方面具有意想不到的作用。

Molecular recognition of ingested nutrients results in the secretion of gastrointestinal hormones regulating digestion and gut motility necessary for absorption of these nutrients. Amino acids, short chain fatty acids, carbohydrates and changes in intraluminal pH are known potent stimulators of gastrin, cholecystokinin and secretin, some of the most important gastrointestinal hormones regulating digestion, secretion and motility. The cells responsible for chemosensation of these nutrients are presumed to be scattered along the mucosal layer lining the gastrointestinal tract and may possibly be neurons within the enteric nervous system. The inherent dispersed nature of these chemosensory cells and the lack of any known markers for their identification create a difficult problem for solving the molecular basis of nutrient recognition. Calcium acting at the calcium sensing receptor on gastric G cells has been shown to stimulate the secretion of gastrin. The CaSR has been shown to be a multimodal sensor for amino aicds and pH in addition to divalent cations. Therefore, we hypothesised that the gastric calcium sensing receptor (CaSR) expressed on antral G cells is the physiologically relevant sensor mediating peptone, amino acids (especially aromatic amino acids) and pH stimulated gastrin release in vivo. This work utilized CaSR and parathyroid hormone (PTH) gene deleted mice provided by Martin R. Pollak (JCI 111:1021-1028, 2003). Plasma gastrin response to peptone, phenylalanine (Phe) and pH was assessed in littermates homozygous deleted (KO) (CaSR- /- /PTH- / -), heterozygous (HET) (CaSR+ /- /PTH- / -) and wild type (WT) (CaSR+ /+/PTH- / -) for the CaSR gene or WT C57/B6 mice. Mice were fasted overnight and either fed ad libitum or gavaged with either peptone (8%), Phe (100 mM), or Hepes (150 mM, pH 7.0) as a bolus or parenteral carbacol (5 mg) or bombesin (50 ug/kg) and the peak gastrin response for each agent was measured by RIA. Gastric acid secretion were measured in response to gastrin -17 (1 mg/kg), histamine (10 mg/kg) or carbachol (5 mg). Hematoxylin and eosin staining and immunohistochemistry was performed on frozen sections from the stomach using rabbit anti-gastrin. We found that CaSR deficient mice do not secrete gastrin in response to intraluminal stimulation with calcium, peptone, L-phenylalanine and elevated pH. However, CaSR deficient mice responded to parenteral bombesin and carbacol similar to WT mice. The gastrin response for ad libitum and gavage feeding in the heterozygous and WT littermates was similar to that observed for WT C57/B6 control mice. High Ca++ dietary correction of plasma Ca++ in the CaSR deficient mice did not restore their gastrin response. CaSR deficient mice expressed a decreased number of antral G cells and gastrin content. They also had an elevated resting gastric pH and reduced basal acid secretion, although they displayed a normal gastric acid secretory response to exogenous gastrin and histamine. From these studies we learned that gastric CaSRs are necessary for the physiological sensing of intraluminal nutrients such as rat chow, peptone, Phe and elevated pH that results in the stimulated release of gastrin from antral G cells. Whether the sensing of these stimulants is directly mediated by CaSRs on gastric antral G cells awaits further studies using isolated G cells. In addition, we found an unexpected role of CaSR in determining antral G cell number.