MOLECULAR MECHANISMS REGULATING CALCIUM FLUX IN SALIVARY GLANDS

调节唾液腺钙通量的分子机制

• 批准号: 5201782
• 负责人: I S AMBUDKAR
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5201782
• 关键词: acinar cell; basolateral membrane; biological fluid transport; calcium flux; interferon gamma; laboratory rat; membrane permeability; parotid gland; salivary glands; secretion; tissue /cell culture

This project is directed towards understanding the processes which regulate cytosolic [Ca] in salivary gland cells. In these cells, Ca entry which is critical for prolonged fluid secretion in this gland, is regulated by a "Ca release-activated Ca entry"-type of mechanism, found in a number of non-excitable cells. This largely uncharacterized mechanism appears to be stimulated by the depletion of Ca in the intracellular Ca store(s). In this reporting period we have demonstrated that Ca influx in rat parotid and HSG cells (derived from the human submandibular gland) is regulated by (i)Ser/Thr phosphorylation (inhibition) and dephosphorylation (stimulation). We had described previously that internal Ca pool-depleted parotid acinar cells have two Ca influx components with high and low affinities for Ca, Kd=65microM and Kd=3.3mM, respectively. In this reporting period we have shown that the high affinity component is more sensitive to inhibition by depolarization and Zn, while both components are inhibited by micronazole and carbodiimide. Further, we have examined the kinetics of Ca influx into isolated basolateral membrane vesicles (BLMV) at a lower temperature (30 degrees C) and have demonstrated the presence of two Ca influx components with Kd similar to that found in intact cells (108microM and 3l.5mM). In the last reporting period we had described reconstitution of the high affinity Ca2+ influx component from BLMV into artificial lipid vesicles, by using a detergent, octylglucoside, dilution method. By using lectin chromatography and the same reconsititution procedure, we have now partially purified this Ca influx component. Continuing our recent studies on the effects of IFN-gamma- and TNF-alpha on the proliferatin and Ca signalling mechanisms in HSG cells, we have now shown that there is a decrease in the SERCA 2 type of CA pumps in IFN-gamma-treated cells. This is a novel observation and likely accounts for the decreased Ca content of internal Ca store(s) in IFN- gamma-treated HSG cells.

该项目是针对理解过程的 调节唾液腺细胞中的胞质[Ca]。 在这些细胞中，CA进入 这对于该腺体的长时间流体分泌至关重要，是 由“ CA释放激活的CA进入” - 机制型调节 许多不可解散的细胞。 这种很大的未表征机制 CA在细胞内CA中的耗竭似乎是受刺激的 商店。 在此报告期内，我们证明了CA涌入 大鼠腮腺和HSG细胞（源自人下颌腺）为 受（I）Ser/Thr磷酸化（抑制）和去磷酸化调节 （刺激）。 我们先前描述了内部CA池耗尽 腮腺腺泡细胞具有两个CA涌入成分，高和低 CA的亲和力分别为65microm和Kd = 3.3mm。 在这个 报告期我们已经表明高亲和力组件更多 对去极化和Zn抑制敏感，而两个组件 被辛唑和碳二二二二二二二二抑制。 此外，我们已经检查了 CA涌入分离的基底外侧膜囊泡的动力学 （BLMV）在较低的温度（30度c）下，已证明 具有KD的两个CA涌入组件的存在类似于在 完整的细胞（108microm和3L.5mm）。 在最后的报告期内 描述了高亲和力Ca2+涌入成分的重建 通过使用清洁剂辛基葡萄糖苷，Blmv进入人造脂质囊泡 稀释法。 通过使用凝集素色谱法和相同 重新尊重程序，我们现在已经部分净化了此CA涌入 成分。 继续我们对IFN-GAMMA和IFN-GAMMA和 在HSG细胞中的增殖和Ca信号传导机制上的TNF-α， 我们现在表明SERCA 2类型的Ca泵有所减少 在IFN-GAMMA处理的细胞中。 这是一个新颖的观察，可能 考虑到IFN-内部CA商店的CA含量降低 γ处理的HSG细胞。