The transfer of lipoprotein-bound cholesterol into adrenal cells was examined. Adrenal glands from unstimulated or corticotropin stimulated hypophysectomized rats were incubated with high density lipoprotein (HDL) or low density lipoprotein LDL containing radiolabeled cholesterol. The rate of transfer of labeled cholesterol from HDL into the glands was two to three times greater than from LDL. Corticotropin stimulation increased the transfer of cholesterol from HDL but not LDL. The effects of corticotropin were not dependent on subsequent cholesterol utilization for steroidogenesis. The process of cholesterol transfer from HDL was linear with time over 2 hr at 370 and greatly reduced at 40. In addition, the transfer process became saturated above an HDL cholesterol concentration of 900 sg/ml. About 25% of the labeled adrenal cholesterol arising from HDL was recovered within the mitochondria. The labeled cholesterol within isolated mitochondria could undergo mitochondrial conversion to pregnenolone. Finally, the delipidated HDL apolipoproteins, apoA-I and apoA-II, when added to incubations containing less than saturating concentrations of HDL, stimulated transfer of labeled cholesterol from HDL to adrenal cells. These studies suggest that rat adrenal tissue possesses an HDL specific hormonally-responsive mechanism for accumulating extracellular cholesterol and that apoA-I and apoA-II have a significant function in the uptake process.The mechanisms responsible for transferring cholesterol from plasma into cells are of particular interest because of their possible role in atherosclerotic disease. Adrenal tissue may serve as a useful model for studying these mechanisms because studies in man (1, 2) and the rat (3, 4) showed that 80% or more of the cholesterol substrate for adrenal steroidogenesis may come from plasma. However, little is known about the process by which extracellular cholesterol enters the adrenal cells. Dexter et al. (4) have observed that the uptake process is stimulated by corticotropin (adrenocorticotrophic hormone, ACTH), and furthermore that the stimulatory effect of ACTH persists even when utilization of accumulating cholesterol is blocked by specific inhibitors.Serum cholesterol is lipoprotein bound, predominantly by the high density lipoprotein (HDL) and low density lipoprotein (LDL) fractions. In the rat (5), 60% of the circulating cholesterol is found in HDL and 30% in LDL, while in man 30% and 60% are found in HDL and LDL, respectively (6). In order to better understand the uptake of cholesterol by adrenal cells as well as ACTH regulation of the uptake process, we have examined the ability of HDL and LDL to serve as substrate for transfer of cholesterol to the adrenal. The observations reported here suggest that HDL is the preferred substrate for adrenal cholesterol uptake, and that uptake from HDL is regulated by ACTH. Furthermore, these studies suggest that the HDL apoproteins, apoA-I and apoA-II, play a role in the cholesterol uptake process. Table legends. Incubations were p...

The rat adrenal cortex possesses at least two mechanisms for uptake of extracellular lipoprotein cholesterol; one is receptor mediated endocytosis and the second is a non-endocytotic pathway. The latter was revealed by the ability of lipoproteins such as HDL which do not possess apo B or apo E to enhance steroid hormone output. Rat adrenocortical cell uptake of HDL cholesterol is saturable and differs for unesterified cholesterol and cholesterol esters. In addition rat adrenocortical cells possess ACTH regulated HDL binding sites although the relationship of HDL receptor activity to cholesterol uptake remains uncertain. Further elucidation of the "HDL pathway" in the rat adrenal cortex may have biologic significance beyond understanding adrenal function since many non-steroidogenic tissues also possess HDL receptors and in man circulating levels of HDL-cholesterol are a strong inverse risk factor for accelerated atherosclerosis.

A B S T R A C T Adrenal ornithiine decarboxylase activity was stimulated in a dose-related manner after administration of ACTH or dibutyryl (6N-2'-O-dibutyryl) cyclic AMP to hypophysectomized rats. Little effect was observed for 2 h, but striking increases in enzyme activity were observed 4 h after administration of these substances. Effects of ACTH and dibutyryl cyclic AMP were not secondary to stimulation of steroidogenesis, since hydrocortisone had no effect on adrenal ornithine decarboxylase although it did stimulate activity of the enzyme in the liver and kidney.ACTH, given subcutaneously to hypophysectomized rats, induced striking increases in adrenal cyclic AMP levels within 15-30 min with a fall towards the base line in 1 h. Increases in ornithine decarboxylase activity lag several hours after this endogenous cyclic AMP peak, in contrast to the stimulation of steroidogenesis by the nucleotide that requires only 2-3 min. After graded doses of ACTH, increases in adrenal cyclic AMP levels at 30 min were paralleled by proportional increases in adrenal ornithine decarboxylase activity 4 h after hormone treatment. Whereas maximal levels of adrenal steroidogenesis have been observed at tissue cyclic AMP levels of 6 nmol/g, ACTH is capable of inducing increases in nucleotide levels up to 200 nmol/g or more. These high tissue levels of cyclic AMP, although unnecessary for maximal steroidogenesis, appear to stimulate adrenal ornithine decarboxylase activity.Several results in addition to the time lag in the stimulation of ornithine decarboxylase activity suggest a mechanism involving accumulation of the enzyme or some factor needed for its activity rather than direct activation of the enzyme by cyclic AMP. Thus, the addition of cyclic AMP directly to the ornithine decarboxylase assay mixture in vitro was without stimulatory Receiz'ed for publication 5 May 1972 and in revised form 27 March 1973. effect. In addition, actinomycin D or cycloheximide in doses sufficient to block adrenal RNA and protein synthesis, respectively inhibited the stimulation of ornithine decarboxylase activity by ACTH in vivo. An adrenocortical cancer was found to maintain ornithine decarboxylase activity at very high levels, but did so at much lower cyclic AMP levels than those of ACTH-stimulated adrenals.It is concluded that ACTH stimulates adrenal ornithine decarboxylase activity and that this effect may be mediated by cyclic AMP. However, cyclic AMP does not appear to be a determinant of the high level of enzyme activity found in adrenocortical cancer.

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Reduced extracellular Ca2+ is known to promote PTH secretion, while severe Mg2+ depletion has the opposite effect. We have correlated the effects of Mg2+ and Ca2+ on parathyroid hormone (PTH) secretion and cAMP accumulation by rat parathyroid tissues in vitro with the effects of these two metals on adenylate cyclase activity in broken membrane preparations. PTH secretion was maximal at 0.5 mM Ca2+, falling to low levels as the Ca2+ concentration was increased to 2.5 mM. Deletion of Mg2+ from the medium resulted in a marked decrease in PTH secretion at any given Ca2+ concentration. At a constant Ca2+ concentration of 1 mM, both PTH secretion and cAMP production rose to maximal rates as the Mg2+ concentration was increased from 0 to 2 mM. The adenylate cyclase of rat parathyroid membranes was stimulated by both GTP and guanyl-5'-yl-imidodiphosphate [Gpp(NH)p]. EDTA-treated membranes could not be stimulated by Gpp(NH)p. Repletion with Mg2+ was more effective than repletion with Ca2+ in restoring responsiveness to the guanine nucleotide. When membranes were maximally preactivated by Gpp(NH)p and then assayed in the presence of variable concentrations of metal ions, enzyme activity was directly inhibited by Ca2+ and stimulated by Mg2+. Adenylate cyclase sensitivity to Ca2+ inhibition was dependent upon the Mg2+ concentration; in the presence of 0.6 mM Mg2+ a 50% inhibition was produced by 0.05 mM Ca2+, while in the presence of 8 mM Mg2+ a 10-fold higher Ca2+ concentration was required for a similar inhibitory effect. The results suggest that Ca2+ may decrease PTH secretion at least in part by a direct inhibition of adenylate cyclase. Mg2+ may promote PTH secretion either by enhancing the activation of adenylate cyclase by endogenous guanine nucleotides or by competing with Ca2+ for binding to a distinct regulatory site on the enzyme.