Crivello J F
Endocrinology. 1985 Aug;117(2):447-56. doi: 10.1210/endo-117-2-447.
The regulation of bovine renal 1 alpha- and 24-hydroxylase activities was examined in primary bovine proximal tubule cell cultures. Maximal 1 alpha- and 24-hydroxylase activities in primary bovine proximal tubule cultures ranged from 1.5-1.8 and 2.0-2.7 pmol/min X 10(6) cells, respectively. The apparent Km was 795 nM for 1 alpha-hydroxylase activity and 1130 nM for 24-hydroxylase activity. 1 alpha- and 24-hydroxylase activities decreased in primary culture after cell plating. Activities decreased both as a function of cell number and as a function of the culture dish. 1 alpha-Hydroxylase activity decayed with a t1/2 of 37 h, while 24-hydroxylase activity decayed with a t1/2 of 45 h. Decreasing cell densities, at which cells were plated, increased the t1/2 for the decay of both activities [t1/2 = 21 h at 5,000 cells/cm vs. t1/2 = 37 h at 25,000 cells/cm for 1 alpha-hydroxylase (P greater than 0.001); t1/2 = 33 h at 5,000 cells/cm vs. t1/2 = 45 h at 25,000 cells/cm for 24-hydroxylase, (P greater than 0.0001)]. Direct addition of 0.25 mM metyrapone inhibited 1 alpha-hydroxylase activity by 33% and 24-hydroxylase activity by 51%. Long term incubation of cell cultures with 0.25 mM metyrapone resulted in a slowing in the loss of both hydroxylase activities, but did not stop the decay. 1 alpha-Hydroxylase activity in 4-day metyrapone-treated cultures was 35% higher than in 4-day untreated cultures. 24-Hydroxylase activity was increased 42% in treated cultures vs. that in untreated cell cultures. Both 1 alpha- and 24-hydroxylase activities were inhibited by direct addition of antioxidants. 1 alpha-Hydroxylase activity was directly inhibited 74% by the addition of 0.1 mM butylated hydroxyanisole (BHA), 69% by the addition of 0.1 mM butylated hydroxytoluene (BHT), and 56% by the addition of 0.05 mM benzyl sulfoxide (BS). 24-Hydroxylase activity was also directly inhibited 72% by 0.1 mM BHA, 55% by 0.1 mM BHT, and 73% 0.05 mM BS. There was no significant difference between the inhibition of either hydroxylase by each antioxidant. Antioxidant mixtures increased the inhibition of hydroxylase activities above that with single antioxidant. The addition of 0.1 mM BHA and 0.05 mM BS to cultures resulted in 100% inhibition of 24-hydroxylase activity and 95% inhibition of 1 alpha-hydroxylase activity. The results were very similar when 0.01 mM BS and 0.1 mM BHT were added to cultures, i.e. 100% and 91% inhibition of 24- and 1 alpha-hydroxylase activities.(ABSTRACT TRUNCATED AT 400 WORDS)
在原代牛近端小管细胞培养物中检测了牛肾1α-羟化酶和24-羟化酶活性的调节。原代牛近端小管培养物中的最大1α-羟化酶和24-羟化酶活性分别为1.5 - 1.8和2.0 - 2.7 pmol/分钟×10⁶个细胞。1α-羟化酶活性的表观Km为795 nM，24-羟化酶活性的表观Km为1130 nM。细胞接种后，原代培养中的1α-羟化酶和24-羟化酶活性下降。活性随细胞数量和培养皿而降低。1α-羟化酶活性以37小时的半衰期衰减，而24-羟化酶活性以45小时的半衰期衰减。接种时细胞密度降低，两种活性衰减的半衰期增加[1α-羟化酶在5000个细胞/cm²时半衰期为21小时，在25000个细胞/cm²时为37小时（P＞0.001）；24-羟化酶在5000个细胞/cm²时半衰期为33小时，在25000个细胞/cm²时为45小时，（P＞0.0001）]。直接添加0.25 mM美替拉酮可抑制1α-羟化酶活性33%，抑制24-羟化酶活性51%。用0.25 mM美替拉酮对细胞培养物进行长期孵育导致两种羟化酶活性丧失减缓，但并未阻止其衰减。在经美替拉酮处理4天的培养物中，1α-羟化酶活性比未经处理4天的培养物高35%。处理后的培养物中24-羟化酶活性比未处理的细胞培养物增加了42%。直接添加抗氧化剂可抑制1α-羟化酶和24-羟化酶活性。添加0.1 mM丁基羟基茴香醚（BHA）可直接抑制1α-羟化酶活性74%，添加0.1 mM丁基羟基甲苯（BHT）可抑制69%，添加0.05 mM苄基亚砜（BS）可抑制56%。0.1 mM BHA也可直接抑制24-羟化酶活性72%，0.1 mM BHT可抑制55%，0.05 mM BS可抑制73%。每种抗氧化剂对两种羟化酶的抑制作用无显著差异。抗氧化剂混合物比单一抗氧化剂对羟化酶活性的抑制作用更强。向培养物中添加0.1 mM BHA和0.05 mM BS可导致2�-羟化酶活性100%抑制，1α-羟化酶活性95%抑制。当向培养物中添加0.01 mM BS和0.1 mM BHT时，结果非常相似，即24-羟化酶和1α-羟化酶活性分别被抑制100%和91%。（摘要截断于400字）
