Gillin A G, Star R A, Sands J M
Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322.
Am J Physiol. 1993 Aug;265(2 Pt 2):F272-7. doi: 10.1152/ajprenal.1993.265.2.F272.
We showed previously that both increasing osmolality by adding NaCl or manitol (hyperosmolarity) or adding vasopressin can stimulate urea permeability in rat terminal inner medullary collecting ducts (IMCD). Vasopressin acts via adenosine 3',5'-cyclic monophosphate (cAMP), but the mechanism by which hyperosmolarity acts is unknown. To study the mechanism, we determined the effect of varying osmolality (with NaCl) on two potential second messenger systems, i.e., cAMP and intracellular calcium. There was no significant difference in cAMP production among tubules incubated at 290, 490, 690, or 890 mosmol/kg. In contrast, cAMP did increase significantly after vasopressin (10(-8) M) addition. Intracellular calcium increased significantly when osmolality was increased from 290 to 490 mosmol/kg in the absence of vasopressin. To examine whether changes in intracellular calcium affect urea permeability, we added thapsigargin (and removed bath calcium) while maintaining osmolality at 290 mosmol/kg. Both intracellular calcium and urea permeability increased significantly. Next, we buffered intracellular calcium by pretreatment with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 50 microM). BAPTA completely blocked the increase in urea permeability occurring when osmolality was increased from 290 to 490 mosmol/kg, but did not block the increase in urea permeability occurring when vasopressin (10(-8) M) was added. In summary, 1) hyperosmolarity increases intracellular calcium, but has no effect on cAMP accumulation; 2) thapsigargin increases intracellular calcium and urea permeability; and 3) BAPTA blocks the hyperosmolarity-stimulated increase in urea permeability, but not vasopressin-stimulated urea permeability.(ABSTRACT TRUNCATED AT 250 WORDS)
我们之前的研究表明，通过添加氯化钠或甘露醇来提高渗透压（高渗）或添加抗利尿激素均可刺激大鼠终末内髓集合管（IMCD）的尿素通透性。抗利尿激素通过3',5'-环磷酸腺苷（cAMP）发挥作用，但高渗作用的机制尚不清楚。为研究该机制，我们测定了不同渗透压（用氯化钠）对两种潜在第二信使系统即cAMP和细胞内钙的影响。在290、490、690或890毫渗摩尔/千克孵育的肾小管中，cAMP生成无显著差异。相比之下，添加抗利尿激素（10⁻⁸ M）后cAMP显著增加。在无抗利尿激素的情况下，当渗透压从290毫渗摩尔/千克增加到490毫渗摩尔/千克时，细胞内钙显著增加。为研究细胞内钙的变化是否影响尿素通透性，我们添加了毒胡萝卜素（并去除浴液中的钙），同时将渗透压维持在290毫渗摩尔/千克。细胞内钙和尿素通透性均显著增加。接下来，我们用1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA，50微摩尔）的乙酰氧甲酯预处理来缓冲细胞内钙。BAPTA完全阻断了渗透压从290毫渗摩尔/千克增加到490毫渗摩尔/千克时尿素通透性的增加，但未阻断添加抗利尿激素（10⁻⁸ M）时尿素通透性的增加。总之，1）高渗增加细胞内钙，但对cAMP积累无影响；2）毒胡萝卜素增加细胞内钙和尿素通透性；3）BAPTA阻断高渗刺激的尿素通透性增加，但不阻断抗利尿激素刺激的尿素通透性增加。（摘要截短于250字）
