Hurley T W, Ryan M P, Brinck R W
Department of Child Health, School of Medicine, University of Missouri-Columbia 65212.
Am J Physiol. 1992 Aug;263(2 Pt 1):C300-7. doi: 10.1152/ajpcell.1992.263.2.C300.
In rat pancreatic and submandibular gland acini during exposure to carbachol, changes in the fluorescence emission intensity ratio (R) of acini loaded with mag-fura-2 resemble changes in cytosolic Ca2+ concentration (Ca2+i) in acini loaded with fura-2. Furthermore, changes of R depend on the presence of extracellular Ca2+ (Ca2+o) but are much less influenced by changes in extracellular Mg2+ (Mg2+o). To evaluate interference with measurement of cytosolic Mg2+ (Mg2+i) by changes in Ca2+i, we determined the dissociation constant (Kd) and Hill coefficient (NH) of the Ca(2+)-mag-fura-2 and Mg(2+)-mag-fura-2 complexes in standard solutions, in intact acini after loading with the acetoxymethyl ester of mag-fura-2 (mag-fura-2/AM), and in lysates derived from acini loaded with mag-fura-2/AM. The Kd of the Ca(2+)-mag-fura-2 complex in acini (determined with ionomycin) was 20.49 +/- 5.20 microM, and NH was 1.44 +/- 0.16 (n = 24). Kd of the Mg(2+)-mag-fura-2 complex in acini was 2.25 +/- 0.98 mM, and NH was 1.20 +/- 0.20 (n = 25). Mean Kd values were slightly lower in acinar lysates and in solutions of standard mag-fura-2. In acini from either gland, 1,2-bis(2-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid (BAPTA) suppressed carbachol-induced Ca2+i transients. The R value in stimulated acini loaded with BAPTA and mag-fura-2 increased slightly when Mg2+o was increased from less than 10 nM to 1.2 mM, suggesting that Mg2+ influx contributes to the maintenance of Mg2+i during exposure to carbachol. Under these conditions, pancreatic acinar Mg2+i is 0.53 +/- 0.14 mM (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)
在大鼠胰腺和下颌下腺腺泡暴露于卡巴胆碱期间,装载了镁-呋喃-2的腺泡的荧光发射强度比值(R)的变化类似于装载了呋喃-2的腺泡中胞质Ca2+浓度(Ca2+i)的变化。此外,R的变化取决于细胞外Ca2+(Ca2+o)的存在,但受细胞外Mg2+(Mg2+o)变化的影响小得多。为了评估Ca2+i变化对胞质Mg2+(Mg2+i)测量的干扰,我们在标准溶液中、用镁-呋喃-2的乙酰氧基甲酯(镁-呋喃-2/AM)装载后的完整腺泡中以及从装载了镁-呋喃-2/AM的腺泡获得的裂解物中,测定了Ca(2+)-镁-呋喃-2和Mg(2+)-镁-呋喃-2复合物的解离常数(Kd)和希尔系数(NH)。腺泡中Ca(2+)-镁-呋喃-2复合物的Kd(用离子霉素测定)为20.49±5.20微摩尔,NH为1.44±0.16(n = 24)。腺泡中Mg(2+)-镁-呋喃-2复合物的Kd为2.25±0.98毫摩尔,NH为1.20±0.20(n = 25)。在腺泡裂解物和标准镁-呋喃-2溶液中的平均Kd值略低。在来自任一腺体的腺泡中,1,2-双(2-氨基苯氧基)乙烷-N,N,N',N'-四乙酸(BAPTA)抑制了卡巴胆碱诱导的Ca2+i瞬变。当Mg2+o从小于10纳摩尔增加到1.2毫摩尔时,装载了BAPTA和镁-呋喃-2的受刺激腺泡中的R值略有增加,这表明在暴露于卡巴胆碱期间Mg2+内流有助于维持Mg2+i。在这些条件下,胰腺腺泡Mg2+i为0.53±0.14毫摩尔(n = 5)。(摘要截断于250字)