Mgbonyebi O P, Smothers C T, Mrotek J J
Physiology Department, Meharry Medical College, Nashville, TN 37208.
Cell Biol Toxicol. 1994 Feb;10(1):23-33. doi: 10.1007/BF00757184.
In previous studies cadmium chloride (CdCl2) nonlethally inhibited Y-1 adrenal mouse adrenal tumour cell 20-dihydroxyprogesterone (20DHP) secretion, affecting unstimulated and stimulated steroidogenic pathway sites differently. We studied CdCl2 effects on unstimulated steroidogenesis using Y-1 cells incubated 0.5 h in medium with or without cadmium (using the concentration that inhibited ACTH-stimulated steroid secretion by 50%). Exogenously added 20-hydroxycholesterol (20OHC), 22(R)-hydroxycholesterol (22OHC), 25-hydroxycholesterol (25OHC), pregnenolone (PREG), or progesterone (PROG) were used to bypass any rate-limited steroidogenic pathway sites that CdCl2 might inhibit. 25OHC is a biologically active nonpathway steroid, while 20OHC, 22OHC, PREG, and PROG are pathway steroids; each increased unstimulated 20DHP secretion nearly 10-fold. Although CdCl2 could not reduce dibutyryl cyclic AMP- (dbcAMP)-stimulated 20DHP secretion significantly, it did significantly reduce basal and 25OHC-induced 20DHP secretion 25% below untreated levels. When 20OHC, 22OHC, PREG, or PROG were incubated with unstimulated Y-1 cells, their synthesis into 20DHP was unaffected by cadmium. dbcAMP bypasses the plasma membrane enzyme complex that synthesizes intracellular cAMP during exogenous ACTH stimulation; dbcAMP was not inhibited by CdCl2. The rate-limited step accelerated by cAMP involves plasma membrane and/or cytoplasmic cholesterol transport to and through outer and inner mitochondrial membranes before the cholesterol is synthesized into pregnenolone by side-chain cleavage enzymes on the inner membrane matrix face. Little is known regarding the mechanisms controlling unstimulated steroidogenesis. Under unstimulated conditions the 25-, 20- and 22(R)-monohydroxyls of cholesterol facilitate plasma membrane, cytoplasm and inner and outer mitochondrial solubility, diffusion and/or transport to bypass rate-limited steps and augment unstimulated steroid synthesis. Since conversion of endogenous mitochondrial cholesterol and 25OHC, but not dbcAMP-mobilized cytoplasmic cholesterol, 20OHC or 22OHC conversion, to 20DHP is inhibited by CdCl2, this suggests that (a) control of mitochondrial cholesterol supplies is independent of the cAMP-regulated mitochondrial steps in the 20DHP steroid synthetic pathway, (b) CdCl2 specifically inhibited endogenous mitochondrial cholesterol and 25OHC utilization, (c) CdCl2 toxicity may affect adrenal, testicular, ovarian, and placental basal steroidogenic functions, and (d) 25OHC may be a useful compound to examine unstimulated steroid synthesis.
在先前的研究中，氯化镉（CdCl₂）以非致死剂量抑制Y-1肾上腺小鼠肾上腺肿瘤细胞分泌20-二羟基孕酮（20DHP），对未受刺激和受刺激的类固醇生成途径位点的影响有所不同。我们使用Y-1细胞在含或不含镉的培养基中孵育0.5小时（使用抑制促肾上腺皮质激素刺激的类固醇分泌50%的浓度），研究了CdCl₂对未受刺激的类固醇生成的影响。外源性添加20-羟基胆固醇（20OHC）、22(R)-羟基胆固醇（22OHC）、25-羟基胆固醇（25OHC）、孕烯醇酮（PREG）或孕酮（PROG），以绕过CdCl₂可能抑制的任何限速类固醇生成途径位点。25OHC是一种生物活性非途径类固醇，而20OHC、22OHC、PREG和PROG是途径类固醇；每种物质都使未受刺激的20DHP分泌增加近10倍。虽然CdCl₂不能显著降低二丁酰环磷腺苷（dbcAMP）刺激的20DHP分泌，但它确实使基础和25OHC诱导的20DHP分泌显著降低至未处理水平以下25%。当20OHC、22OHC、PREG或PROG与未受刺激的Y-1细胞一起孵育时，它们合成20DHP的过程不受镉的影响。dbcAMP在外源性促肾上腺皮质激素刺激期间绕过合成细胞内cAMP的质膜酶复合物；CdCl₂不抑制dbcAMP。cAMP加速的限速步骤涉及质膜和/或细胞质胆固醇向线粒体外膜和内膜的转运以及通过线粒体外膜和内膜的转运，然后胆固醇在内膜基质表面被侧链裂解酶合成为孕烯醇酮。关于控制未受刺激的类固醇生成的机制知之甚少。在未受刺激的条件下，胆固醇的25-、20-和22(R)-单羟基促进质膜、细胞质以及线粒体内外膜的溶解性、扩散和/或转运，以绕过限速步骤并增加未受刺激的类固醇合成。由于内源性线粒体胆固醇和25OHC转化为20DHP受到CdCl₂的抑制，但dbcAMP动员的细胞质胆固醇、20OHC或22OHC的转化不受影响，这表明（a）线粒体胆固醇供应的控制独立于20DHP类固醇合成途径中cAMP调节的线粒体步骤，（b）CdCl₂特异性抑制内源性线粒体胆固醇和25OHC的利用，（c）CdCl₂毒性可能影响肾上腺、睾丸、卵巢和胎盘的基础类固醇生成功能，以及（d）25OHC可能是一种用于研究未受刺激的类固醇合成的有用化合物。
