Regulation of low-density lipoprotein receptors in cultured bovine adrenocortical cells

Bovine adrenocortical cells in monolayer culture produce cortisol and respond to corticotropin (ACTH) by an increase in cortisol secretion. Several lines of evidence are indicative that much of the cholesterol that serves as precursor for steroid hormone biosynthesis by these cells is derived from low-density lipoprotein (LDL) cholesterol that is taken up endocytotically by means of specific receptors localized in bovine adrenocortical plasma membranes. ACTH stimulated this process concomitant with an increase in steroid production. In the absence of LDL, ACTH had no effect on steroid biosynthesis. ACTH action in bovine adrenocortical cells resulted in an increase in the number of LDL receptor sites in the membrane fractions, whereas the dissociation constant for LDL binding was not changed. Chloroquine and NH₄Cl, considered to be inhibitors of lysosomal degradative activity, caused an increase in the number of [¹²⁵I]iodoLDL binding sites in the plasma membrane but the effect of ACTH was still apparent in the presence of these agents. These results are suggestive that the lifetime of the LDL receptor is increased when lysosomal activity is inhibited. When aminoglutethimide was added to block cholesterol side-chain cleavage activity and inhibit steroid production, the number of [¹²⁵I]iodoLDL binding sites in the membrane fractions prepared from bovine adrenocortical cells cultured in the presence of ACTH was reduced to 50% of that in cells maintained in aminoglutethimide-free medium. However, under these conditions the number of binding sites was still significantly greater than in cells maintained in the absence of ACTH. The effects of aminoglutethimide on uptake and degradation of [¹²⁵I]iodoLDL were similar to the effects on the number of [¹²⁵I]iodoLDL binding sites. Based on these results, we conclude that the action of ACTH to stimulate LDL metabolism in bovine adrenocortical cells results from an increase in the number of LDL binding sites in the plasma membranes. This action of ACTH appears to be, at least in part, independent of cholesterol utilization for cortisol biosynthesis. However, the effect of aminoglutethimide is indicative that changes in the intracellular cholesterol concentration might modulate the action of ACTH to increase the number of LDL binding sites and therefore to stimulate LDL degradation.     

Biological Activity of Fractions Obtained by Differential Centrifugation of the Bovine Adrenal Cortex

This study is an attempt to correlate adrenocortical hormonal activity with various cell fractions obtained by homogenization and differential centrifugation of bovine adrenocortical tissue. Bioassays involving the decrease in circulating eosinophils and sodium retention were employed for the estimation of the glucocorticoids and mineralocorticoids respectively. The supernatant was the most active fraction in both bioassays, while the microsome and mitochondrial fractions demonstrated varying degrees of activity. The total nitrogen, lipide phosphorus, and cholesterol contents of the fractions were determined. The microsome fraction contained a relatively higher amount of lipide phosphorus and a marked amount of cholesterol.     

Lipoprotein receptors and steroidogenesis in adrenocortical cells.

Steroid-producing tissues require a continuous supply of cholesterol for hormone synthesis. In the majority of the steroidogenic tissues the cholesterol is imported via the receptor-mediated uptake of lipoproteins, and therefore the influence on the lipoprotein receptors provides an additional level for the regulation of hormone synthesis. Hormones regulating the adrenocortical activity exert both short- and long-term action, and thus they may control the interactions of the major cholesterol delivery particles--low- (LDLs) and high-density lipoproteins (HDLs)--and their receptors in short- and long-term action, possibly modulating the signal transduction in the former case and the number and distribution in the latter. The LDL and HDL pathway and the signal transduction mechanism is briefly reviewed. Data are discussed concerning short- and long-term action of hormones (alpha-MSH and ACTH, respectively) on the HDL3 receptors of isolated adrenocortical cells. Short-term treatment with alpha-MSH and long-term treatment with ACTH increased the binding of HDL3 to zona glomerulosa and fasciculata cells, respectively, while both treatments increased the hormone production in the presence of HDL. The lipoprotein receptors were frequently found on the microvilli of adrenocortical cell membranes.     

The effects of taxol, a microtubule-stabilizing drug, on steroidogenic cells

The effects of taxol on steroid production and microtubule polymerization were examined using Y-1 adrenocortical tumor cells, MLTC-1 Leydig tumor cells, and primary cultures of bovine adrenocortical cells. Taxol inhibited the following steroidogenic processes within the Y-1 and MLTC-1 cells: (1) hormonal increase of steroid production, (2) dibutyryl cyclic AMP-increased steroid production, and (3) hormone-stimulated pregnenolone production. The inhibitory action of taxol was concentration dependent and also resulted in an increase in cytoplasmic microtubules. In addition, the inhibitory action of taxol on hormone-stimulated steroid production was reversible. Taxol appeared to inhibit cholesterol movement to the mitochondrial site of cholesterol side-chain cleavage enzyme but did not affect overall protein synthesis. Interestingly, taxol did not affect hormone-stimulated steroid production in bovine adrenocortical cells. This lack of inhibition may correspond to the ultrastructural observation that microtubule bundling after taxol treatment was observed in the tumor cells but not in similarly treated bovine adrenal cells. With this conflicting information between cell types, a direct relationship between taxol treatment and inhibition of steroid production has not been established. However, these results suggest that taxol alters the rate of transport of cholesterol to the cholesterol side-chain cleavage enzyme within the steroidogenic tumor cells.     

Cholinergic muscarinic stimulation of steroidogenesis in bovine adrenal cortex fasciculata cell suspensions

Acetylcholine was found t acutely stimulate cortisol production by bovine fasciculata adrenocortical cell suspensions. This effect was maximal at 10^?4 M acetylcholine concentration, resulted in a 5-fold increase in cortisol production over the control after 1 h incubation, and represented about one fifth of the ACTH maximal stimulation under the same conditions. Acetylcholine-stimulated steroidogenesis was concentration-dependent (10^?8–10^?5 M), propotional to the cell numbe (5 · 10⁵–2 · 10⁶) and reached a plateau after 30 min incubation. Use of various cholinergic specific agonists and antagonists showed that thet steroidogenic action of acetylcholine was a typical muscarinic effect. This character is in agreement with the previously demonstrated presence of muscarinic receptors in bovine adrenocortical tissue. The steroidogenic effect of acetylcholine required the presence of extracellular calcium in the medium and was impaired upon addition of tetracaine and procaine. No change in cyclic AMP nor cyclic GMP levels could be detected in the system under acetylcholine stimulation. Acetylcholine appeared to exhibit a synergistic in combination with ACTH, and exogenous cyclic AMP; these observations suggest a different mechanism of action for acetylcholine and ACTH and point to a possible cholinergic participation in the regulation of adrenocortical differentiated functions in vivo.     

Lipoprotein lipase promoting agent, NO-1886, modulates adrenal functions: species difference in effects of NO-1886 on steroidogenesis.

A novel compound, NO-1886, which possesses a powerful lipoprotein lipase activity-increasing action, induces hypertrophy of adrenals in rats and hyperplasia of cortical cells in dogs. However, these effects were not observed in monkeys. We examined the effects of NO- 1886 on steroid hormone production by adrenocortical cells to clarify its effects on adrenal steroidogenesis. NO-1886 did not inhibit the steroid synthetic enzymes, including 3beta-hydroxysteroid dehydrogenase, 21-hydroxylase, 11beta-hydroxylase, or cholesterol side-chain cleavage enzymes. However, NO-1886 affected steroid production from adrenocortical cells in rats, dogs, monkeys, and humans in in vitro studies. These effects were almost completely reversed by the addition of 25-hydroxycholesterol or low-density lipoproteins to the reaction medium, but not reversed by the addition of high-density lipoproteins. These results suggest that NO-1886 affects the cholesterol pathways within the adrenocortical cells and inhibits steroidogenesis, causing a reduction of steroid hormone release from adrenocortical cells and resulting in hypertrophy of adrenals via feed-back mechanisms. However, its effect is not apparent in animals that use low-density lipoproteins as a source of adrenocortical steroidogenesis.     

Histochemical studies of the adrenocortical homologue in the kidney of the Australian lungfish,Neoceratodus forsteri

A histochemical study has been made of groups of cells lying adjacent to the blood vessels of the kidney of Neoceratodus forsteri. The cells contain lipid droplets of varying sizes, stain positively for cholesterol and its esters and contain 3betaol-steroid dehydrogenase activity. Similar cells have previously been described in another dipnoan fish, Protopterus, and in a urodele, Pleurodeles waltlii and are considered to be the adrenocortical tissue of these species. Because of the histochemical characteristics of the cells together with their location, we conclude that they represent the adrenocortical homologue of N. forsteri. As is the case in Protopterus, no chromaffin tissue was detected in the kidney of N. forsteri.     

The role of mitochondrial cytochrome P-450 from bovine adrenal cortex in side chain cleavage of 20S,22R-dihydroxycholesterol.

The role of cytochrome P-450 in the side chain cleavage of 20S,22R-dihydroxycholesterol was investigated by examining the effect of carbon monoxide on the conversion of this substance to pregnenolone by cytochrome P-450 from bovine adrenocortical mitochondria; the effect of carbon monoxide on the conversion of cholesterol to pregnenolone by the same enzyme also was examined. Fifty per cent inhibition of side chain cleavage was produced by gas mixtures with the following ratios: CO:O2,1.5 for cholesterol and 1.2 for 20S, 22R-dihydroxycholesterol. Photochemical action spectra revealed that light of wavelength 451 nm decreased the inhibition of side chain cleavage of both substrates to a greater extent than light of other wavelenghts. It is concluded that the heme moiety of P-450 is involved in the cleavage of 20S,22R-dihydroxycholesterol.     

Studies on lipoprotein and adrenal steroidogenesis: I. Roles of low density lipoprotein- and high density lipoprotein-cholesterol in steroid production in cultured human adrenocortical cells

The roles of human low density lipoprotein (LDL)- cholesterol and high density lipoprotein (HDL)- cholesterol on adrenal steroidogenesis were investigated using cultured human adult and fetal adrenocortical cells and the findings were then compared to those obtained with bovine adrenocortical cells. The secretion of cortisol in both human and bovine adrenocortical cells was dose-dependently increased by the administration of LDL- or HDL-cholesterol in the presence of adrenocorticotropin (ACTH). LDL-cholesterol was utilized to a greater extent than HDL-cholesterol in both human and bovine adrenal steroidogenesis in the presence of ACTH. Exogenous lipoprotein-derived cholesterol was less utilized in human adrenal steroidogenesis than in bovine adrenal steroidogenesis, compared to the endogenous cholesterol. An increase in the secretion of cortisol and dehydroepi androsterone sulfate (DHEA-S) continued for the 5-day culture period, in the presence of lipoprotein cholesterol and ACTH in both human adult and fetal adrenocortical cells. The secretion of aldosterone increased on the first day of the culture period, then gradually decreased for the 5-day culture period in human adult adrenocortical cells, but not in human fetal adrenocortical cells in the presence of lipoprotein cholesterol and ACTH. These findings demonstrate that exogenous cholesterol utilized in the biosynthesis of steroids is mainly from LDL-cholesterol in both human adult and fetal adrenals and bovine adrenal and the proportion of cholesterol synthesized de novo is significantly larger in the human adult adrenal than in the bovine adrenal.     

Adrenocortical function in 4-APP treated rats: a coupled stereological and biochemical study

Adrenocortical function in 4-APP-induced (4-aminopyrazolo[3,4-d]pyrymidine) lipoprotein-deficient rats was studied in relation to quantitative morphologic changes in the gland. 4-APP treatment results in enlargement of the adrenal cortex and its zona fasciculata and reticularis cells. In enlarged livers, cholesterol and free fatty acid concentrations were similar to that of control rats, however a marked accumulation of triglycerides with a concomitant drop in hepatic delta 4-steroid hydrogenase activity was found. A profound drop in serum cholesterol in both, high and low density lipoproteins, as well as triglycerides and plasma corticosterone concentrations was accompanied by a marked lowering of cholesterol and corticosterone concentration in the adrenal gland. Corticosterone output by adrenal homogenates was higher in 4-APP treated rats than in control animals. Such a treatment did not change cholesterol side-chain cleavage, 11 beta-hydroxylase, 3 beta-ol dehydrogenase-isomerase, steroid 5 alpha-reductase and neutral lipase activities when expressing results per unit weight of tissue or protein. However, when calculating per adrenocortical cell, adenine analogue applied increased 11 beta-hydroxylase, steroid 5 alpha-reductase and neutral lipase activities. Thus, coupled biochemical and stereologic studies revealed a complex and multidirectional effect of 4-APP on the rat adrenal cortex. This effect may be caused by serum lipoprotein deficiency and by toxic and stressful action of the adenine analogue on the rat. Also a direct effect of 4-APP on rat adrenal cortex may not be excluded.     

Low-density lipoprotein receptor activity in the guinea pig adrenal cortex. II. Zonal response to aminoglutethimide and 17 alpha-ethinylestradiol

Low-density lipoprotein (LDL) receptor activity and the concentration of cholesterol were measured in the outer (glomerulosa/fasciculata) and inner (reticularis) zones of the adrenal cortex of the guinea pig to examine the relation between cholesterol content and LDL receptor activity. While the concentration of cholesterol was 2-3-times higher in the outer cortical zone, the maximum high-affinity binding capacity for LDL was essentially the same for the two zones, or slightly higher for the inner zone. Adrenocorticotrophic hormone (ACTH) caused a significant increase in LDL receptor activity only in the outer zone, but led to a reduction in the cholesterol content in both adrenocortical zones. The treatment of animals with 17 alpha-ethinyl-estradiol also resulted in a reduction of cholesterol in both adrenocortical zones, but an increase in LDL receptor number only in the outer zone. The latter effect was partially reversed by the administration of dexamethasone. Aminoglutethimide, which was used in a dose that did not block steroidogenesis but did block the hydrolysis of cholesteryl esters in response to ACTH, did not prevent the ACTH-induced increase in LDL receptor number in the outer zone. Thus, the number of LDL receptors was increased in the zona fasciculata by ACTH in the absence of a reduction in cellular cholesterol content, while the number of LDL receptors in the zona reticularis was not increased by ACTH even in the face of a reduction in cellular cholesterol. Exclusive of the experiments employing aminoglutethimide, when the cellular cholesterol content was plotted against LDL binding activity, an excellent inverse correlation was revealed for the zona fasciculata, but essentially no correlation was noted for the zona reticularis. It is concluded that the outer and inner cortical zones of the guinea pig adrenal are quite distinct in the nature of their LDL receptor activity and regulation: the LDL receptor of the outer zone appears to function in a way similar to what has been reported for the whole adrenal cortex of other species in that receptor number correlates with tissue cholesterol content and is primarily regulated by ACTH; the LDL receptor number of the inner zone, however, does not correlate with tissue cholesterol content and is apparently not regulated by ACTH.     

Peripheral-type benzodiazepine receptors are involved in the regulation of cholesterol side chain cleavage in adrenocortical mitochondria

In an attempt to elucidate the physiological relevance of the peripheral type of benzodiazepine receptor in adrenocortical mitochondria, we examined the effect of three different benzodiazepines (diazepam, Ro5-4864, and chlordiazepoxide) on the conversion of cholesterol to pregnenolone, the rate-limiting step in steroidogenesis, by using cholesterol-loaded mitochondria from bovine adrenal zona fasciculata. These benzodiazepines, except chlordiazepoxide, caused a dose-dependent stimulation of the cholesterol side chain cleavage in the mitochondria. The stimulatory effect of Ro5-4864 was approximately 10 times more potent than that of diazepam. No inhibitory effect of YM-684 (Ro15-1788), a potent antagonist to central-type benzodiazepine receptors, was observed in the stimulation induced by diazepam and Ro5-4864. Both external calcium ion and voltage-dependent calcium channel blocker, (+)-PN200-110, were without effect on the diazepam-induced steroidogenesis. By contrast, pretreatment of mitochondria with digitonin abolished the stimulatory effect of diazepam on the mitochondrial steroidogenesis. The present results indicate that the peripheral-type benzodiazepine receptor of adrenocortical mitochondria plays an essential role in regulating cholesterol side chain cleavage without any change of calcium channels.     

Effects of prolonged ACTH-stimulation on adrenocortical accumulation of lipofuscin granules in aged rats

Subcellular deposition of lipofuscin granules is a marker of aging. Human and rodent adrenal cortices accumulate lipofuscin granules with age, but the mechanism that leads to the accumulation is not known. The ultrastructural appearance of lipofuscin granules resembles that of secondary lysosomes. Since adrenocortical subcellular events are predominantly influenced by ACTH action, we therefore studied the effect of prolonged ACTH-stimulation on adrenocortical accumulation of secondary lysosome-like granules, designated herein as lipofuscin granules. Using aged Fischer 344 male rats as a model, we found that a 7 day ACTH stimulation exerts a reducing effect on adrenocortical lipofuscin accumulation. Thus, adrenocortical accumulation of lipofuscin granules with age in vivo may not be an irreversible process.     

Varying the duration of A23187 administration alters its effect on adrenal steroidogenesis

A variety of effects of A23187 have been reported as its actions on adrenocortical steroidogenesis. This diversity probably resulted because of differences in the protocol of applying the Ca++ ionophore. We continue to observe a dose-dependent potentiation by the ionophore on ACTH-stimulated corticosterone secretory activity of superfused rat adrenocortical slices. This effect was eliminated or reversed if the tissue was pretreated with A23187 for 30 min prior to secretagogue application. The quality of the ionophore effect also depends on the submaximal dose of ACTH employed.     

ACTH induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase, cholesterol biosynthesis, and steroidogenesis in primary cultures of bovine adrenocortical cells

The current studies demonstrate that corticosteroidogenesis can be maintained by primary cultures of bovine adrenocortical cells under lipoprotein-depleted conditions. The cholesterol necessary as substrate for steroid synthesis was found to arise from de novo synthesis within these cells. Adrenocorticotropin (ACTH) increased 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity 5-fold within 12 h after addition to the medium. The increase in activity apparently represented accumulation of enzyme as determined by protein blotting and immunodetection. The predominant immunodetectable species of HMG-CoA reductase from bovine adrenal cells was 97,000 daltons; no higher molecular mass species was detectable. The ACTH induction of HMG-CoA reductase activity could be prevented after inhibition of cholesterol conversion to pregnenolone with clotrimazole. These results are suggestive that ACTH increases adrenocortical cholesterol biosynthesis and HMG-CoA reductase activity after conversion of a cellular pool of cholesterol and/or oxysterol into steroid. The increased rate of cholesterol biosynthesis is then capable of maintaining ACTH-promoted steroid production. This is the first study, in vitro, to demonstrate an ACTH-promoted accumulation of HMG-CoA reductase of adrenocortical cells.     

Adrenocortical function in type II hyperlipoproteinemic patients treated with lovastatin (mevinolin)

Lovastatin (Mevinolin), a competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase, has been used effectively as a hypocholesterolemic agent in man. As an inhibitor of endogenous cholesterol synthesis, a potentially serious side effect of therapy with this drug is interference with adrenocortical function. The effect of lovastatin on adrenal function was evaluated in a 6-month, randomized, double blinded, placebo-controlled, crossover study involving 24 type II hyperlipoproteinemic patients. Despite significant lowering of total and low density lipoprotein (LDL) cholesterol by lovastatin, no statistically or clinically significant differences were seen in free cortisol excretion or in plasma cortisol response to intravenous ACTH infusion between baseline, placebo, and lovastatin-treated patients. We conclude that lovastatin does not adversely affect adrenocortical reserve in patients with heterozygous familial hypercholesterolemia (FH) or non-FH type II hyperlipoproteinemia.     

Effects of endogenous and exogenous cholesterol on the ultrastructure and steroid secretion of undifferentiated rat adrenocortical cells in primary culture

Summary The present study was undertaken to define the effects of lipoprotein-derived cholesterol and endogenous, de novo synthesized cholesterol on the ultrastructure and function of undifferentiated rat adrenocortical cells [lipoprotein (HDL₃ and LDL) receptor-negative, zona glomerulosa-like adrenocortical cells] in primary culture. For this purpose human plasma high density lipoprotein (HDL₃) or low density lipoprotein (LDL) was added to culture medium devoid of cholesterol. Steroid secretion remained at the low basal level even after addition of lipoproteins, and the amount of intracellular lipid droplets did not increase. When mevinolin (0.96 µg/ml), an inhibitor of cholesterol synthesis, was added to the culture medium, a low secretion of corticosterone was measured both in serum-free and serum-containing media. Ultrastructurally, lipid droplets disappeared after treatment with mevinolin in both media used. At this concentration of mevinolin cell proliferation was similar to that in the controls, but at higher concentrations (4.8 or 9.6 µg/ml) proliferation was inhibited to 42% and 26% in serum-free medium, and 20% and 12% in serum-supplemented medium, respectively. This study demonstrates that cell proliferation and synthesis of corticosterone by undifferentiated rat adrenocortical cells is identical in the absence or presence of exogenous lipoprotein cholesterol. Inhibition of de novo cholesterol synthesis by mevinolin over a period of 7 days does not inhibit corticosterone secretion or proliferation of cells but decreases the amount of intracellular lipid droplets, thus suggesting utilization of intracellular cholesterol esters. However, higher concentrations of mevinolin inhibit proliferation of cells both in serum-free and serum-containing media.     

Diabetic lipoproteins and adrenal aldosterone synthesis--a possible pathophysiological link?

An increased prevalence of diabetes mellitus (DM) has been reported in patients with primary aldosteronism (PA). DM is associated with abnormal structure and metabolism of circulating lipoproteins, which normally serve as a major source of cholesterol for adrenocortical steroidogenesis. The present study has been designed to investigate the effect of diabetically modified lipoproteins on adrenocortical aldosterone synthesis. Lipoproteins (VLDL, LDL, HDL) isolated from healthy volunteers, were subjected to oxidation or glycoxidation in the presence of sodium hypochlorite (3 mmol/l) or glucose (200 mmol/l), and aldosterone synthesis in human adrenocortical cells (H295R) was examined. Native and glycoxidized VLDL had greatest stimulatory effect on aldosterone production by 15-fold and 14-fold, respectively. At the molecular level, these VLDL produced maximum increases in Cyp11B2 mRNA level up to 17-fold. Experiments with the highly selective scavenger receptor class B type I (SR-BI) inhibitor BLT-1 revealed that cholesterol uptake from native and glycoxidized HDL and VLDL for hormone production is considerably mediated by SR-BI. Western blot analysis of extracellular signal-regulated kinase (ERK 1/2) phosphorylation and experiments with the MEK inhibitor U0126 indicated a specific mechanistic role of the ERK cascade in lipoprotein-mediated steroid hormone release. In summary, diabetic dyslipidemia and modification of circulating lipoproteins may promote adrenocortical aldosterone synthesis.     

Action of vasoactive intestinal peptide (VIP) on amphibian adrenocortical function,in vitro

Vasoactive intestinal peptide (VIP) is located in chromaffin cells of the frog adrenal gland and is able to stimulate corticosteroid secretion in amphibians. In the present study we have investigated the possible involvement of prostaglandins, microfilaments and calcium in the mechanism of action of VIP on frog adrenocortical tissue. Rana ridibunda interrenal dice were perifused with amphibian culture medium for more than 10 hours. Corticosterone and aldosterone concentrations were measured in the effluent perifusate using sensitive and specific radioimmunoassay methods. In the presence of indomethacin (5 μM), a specific blocker of prostaglandin biosynthesis, the spontaneous secretion of corticosteroids was markedly reduced (80%) but the stimulatory effect of VIP was not altered. The administration of the microfilament disrupting agent cytochalasin B (50 μM) inhibited both spontaneous and VIP-induced corticosteroid secretion. In the absence of calcium, the spontaneous level of corticosteroid was reduced to about 60% but VIP was still able to stimulate corticosteroid secretion. From these data we conclude that the integrity of the cytoskeleton is required for the secretory response of adrenocortical cells to VIP, whereas neither prostaglandins nor calcium are involved in VIP-induced adrenocortical stimulation.