Shibata H, Ghishan F K
Department of Pediatrics and Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232.
Pediatr Res. 1990 Dec;28(6):591-4. doi: 10.1203/00006450-199012000-00009.
Our studies were designed to characterize calcium transport by intestinal Golgi vesicles in spontaneously hypertensive rats (SHR) and their genetically matched control, Wistar-Kyoto rats (WKY). The biochemical purity of the intestinal Golgi in SHR and WKY was validated by marker enzyme studies. Calcium uptake by Golgi vesicles represented transport into the intravesicular space as evidenced by temperature dependency and by calcium ionophore A23187-induced calcium efflux experiments. ATP-driven calcium uptake was stimulated several-fold compared with uptake in the absence of ATP and adenylyl-(beta-gamma-methylendiphosphate) (nonhydrolyzable ATP) in both SHR and WKY. ATP-dependent calcium uptake was significantly higher in WKY compared with SHR at early times points, 15 s-5 min (p less than 0.05-0.01). The initial rate of calcium uptake was linear up to 60 s. Kinetic parameters of calcium uptake at free calcium concentrations of 0.1 to 2.0 microM showed a Vmax of 1.64 +/- 0.06 and 1.2 +/- 0.06 nmol.mg protein-1.15 s-1 in WKY and SHR, respectively (p less than 0.01), and the Km values were 0.17 +/- 0.03 and 0.16 +/- 0.04 microM, respectively. Kinetic analysis of ATP-dependent calcium uptake in 3-wk-old rats showed a Vmax of 0.07 +/- 0.005 and 0.36 +/- 0.05 nmol/mg protein-1.15 s-1 (p less than 0.01) and a Km of 0.26 +/- 0.08 and 0.4 +/- 0.2 microM in SHR and WKY, respectively. These results suggest that intestinal Golgi vesicles in SHR and WKY demonstrate an ATP-driven calcium uptake. This ATP-dependent process is significantly decreased in the weanling and adult SHR compared with WKY. Such an abnormality in intracellular calcium regulation may have a role in the development of hypertension.
我们的研究旨在表征自发性高血压大鼠（SHR）及其基因匹配的对照Wistar-Kyoto大鼠（WKY）肠道高尔基体囊泡的钙转运情况。通过标记酶研究验证了SHR和WKY中肠道高尔基体的生化纯度。高尔基体囊泡对钙的摄取代表其转运至囊泡内空间，这一点通过温度依赖性以及钙离子载体A23187诱导的钙外流实验得以证明。与在无ATP和腺苷 -（β - γ - 亚甲基二磷酸）（不可水解ATP）情况下的摄取相比，ATP驱动的钙摄取在SHR和WKY中均增加了数倍。在早期时间点（15秒至5分钟），WKY中依赖ATP的钙摄取显著高于SHR（p小于0.05至0.01）。钙摄取的初始速率在60秒内呈线性。在游离钙浓度为0.1至2.0微摩尔时，WKY和SHR中钙摄取的动力学参数显示，Vmax分别为1.64±0.06和1.2±0.06纳摩尔·毫克蛋白⁻¹·15秒⁻¹（p小于0.01），Km值分别为0.17±0.03和0.16±0.04微摩尔。对3周龄大鼠依赖ATP的钙摄取进行动力学分析显示，SHR和WKY中的Vmax分别为0.07±0.005和0.36±0.05纳摩尔/毫克蛋白⁻¹·15秒⁻¹（p小于0.01），Km分别为0.26±0.08和0.4±0.2微摩尔。这些结果表明，SHR和WKY中的肠道高尔基体囊泡表现出ATP驱动的钙摄取。与WKY相比，断奶和成年SHR中这种依赖ATP的过程显著降低。细胞内钙调节的这种异常可能在高血压的发展中起作用。
