Arachidonate metabolism and the signaling pathway of induction of apoptosis by oxidized LDL/oxysterol.

Owing at least in part to oxysterol components that can induce apoptosis, oxidized LDL (oxLDL) is cytotoxic to mammalian cells with receptors that can internalize it. Vascular cells possess such receptors, and it appears that the apoptotic response of vascular cells to the oxysterols borne by oxLDL is an important part of the atherogenic effects of oxLDL. Thus, an analysis of the signaling pathway of apoptotic induction by oxysterols is of value in understanding the development of atherosclerotic plaque. In a prior study, we demonstrated an induction of calcium ion flux into cells treated with 25-hydroxycholesterol (25-OHC) and showed that this response is essential for 25-OHC-induced apoptosis. One possible signal transduction pathway initiated by calcium ion fluxes is the activation of cytosolic phospholipase A2 (cPLA2). In the current study, we demonstrate that activation of cPLA2 does occur in both macrophages and fibroblasts treated with 25-OHC or oxLDL. Activation is evidenced by 25-OHC-induced relocalization of cPLA2 to the nuclear envelope and arachidonic acid release. Loss of cPLA2 activity, either through genetic knockout in mice, or by treatment with a cPLA2 inhibitor, results in an attenuation of arachidonic acid release as well as of the apoptotic response to oxLDL in peritoneal macrophages or to 25-OHC in cultured fibroblast and macrophage cell lines.

Mechanisms of oxysterol-induced apoptosis.

The rationale for the present review is that oxysterols found in oxidized LDL (oxLDL) play a role in atherogenesis. This perspective is based on studies that show that induction of apoptosis in vascular cells is an important process in atherogenesis, that apoptosis can be induced by oxLDL, and that the oxysterol component of oxLDL is responsible for its proapoptotic activity. The evidence for these concepts is reviewed, as are studies on the mechanisms by which oxysterols can induce apoptosis. An elevation in intracellular calcium appears to be an early signal transduction event that leads to apoptosis through both the extrinsic and intrinsic apoptotic pathways.

Isolation of a somatic cell mutant resistant to the induction of apoptosis by oxidized low density lipoprotein.

Oxidized low density lipoprotein (oxLDL) induces apoptosis in macrophages, smooth muscle cells, and endothelial cells. To elucidate the molecular mechanism of oxLDL-induced cytotoxicity and determine its tissue specificity, we have used Chinese hamster ovary (CHO)-K1 cells expressing human CD36 (CHO/CD36). Expression of CD36 rendered these cells susceptible to killing by oxLDL. This cytotoxicity was due to the induction of apoptosis. Therefore, CD36 expression is the only requirement for oxLDL-induced apoptosis. Oxysterols apparently mediate the cytotoxicity of oxLDL in macrophage foam cells and endothelial cells. 25-Hydroxycholesterol, at concentrations higher than 1 microg/ml, killed CHO-K1 cells, by apoptosis, in medium supplemented with serum as a source of cholesterol. These effects were not seen in a 25-hydroxycholesterol-resistant CHO/CD36 mutant (OX(R)), which was otherwise capable of undergoing apoptosis in response to staurosporine. This mutant was also resistant to killing by oxLDL, suggesting that oxysterols are at least partially responsible for the toxic effects of oxLDL. Oxysterol-induced apoptosis did not involve regulation of sterol regulatory element-binding protein proteolysis or the cholesterol biosynthetic pathway. 25-Hydroxycholesterol stimulated calcium uptake by CHO-K1 cells within 2 min after addition. Treatment of CHO or THP-1 (macrophage) cells with the calcium channel blocker nifedipine prevented 25-hydroxycholesterol induction of apoptosis. OX(R) showed no enhanced calcium uptake in response to 25-hydroxycholesterol.

Oleate potentiates oxysterol inhibition of transcription from sterol regulatory element-1-regulated promoters and maturation of sterol regulatory element-binding proteins.

Activation of genes containing SRE-1 (sterol regulatory element 1) sequences is known to be under the regulation of sterols through modulation of the proteolytic maturation of SREBPs (SRE-1-binding proteins). Previous work has demonstrated SREBP-mediated transcriptional activation of genes encoding enzymes of sterol and fatty acid biosynthesis. Because synthesis of both sterols and C18 fatty acids are required for cell growth, in the absence of exogenous supplements of these lipids, we examined the hypothesis that fatty acid can also be regulatory in SREBP maturation. Our data indicate that C18 fatty acids can potentiate the biological activities of a typical, regulatory sterol: 25-hydroxycholesterol. Inhibition of C18 fatty acid synthesis in cells cultured in serum-free medium renders them resistant to killing by 25-hydroxycholesterol. Repression of expression of reporter constructs driven by promoters bearing SRE-1 element(s) by 25-hydroxycholesterol is increased by C18 fatty acid supplementation. C18 fatty acids also increase the inhibitory effect of 25-hydroxycholesterol on proteolytic maturation and nuclear localization of SREBPs. Furthermore, we also show that C18 fatty acid supplementation can enhance the inhibitory effect of 25-hydroxycholesterol on sterol and fatty acid biosynthesis. These results demonstrate that maximal down-regulation of SREBP maturation and the consequent repression of SRE-1 promoters occurs in response to both a regulatory sterol and fatty acid.

Progesterone stimulation of HMG-CoA reductase activity in cultured cells.

In a previous study we showed that progesterone (PG) stimulated HMG-CoA reductase (HMGR) activity in rat intestinal epithelial cells (IEC-6) incubated in the presence or absence of low-density lipoprotein (LDL) [1,2]. In the present study we examined further the mechanism of this stimulation. We observed that the stimulation of HMGR activity by PG was completely prevented by cycloheximide. Turnover studies utilizing immunoprecipitation of HMGR-labeled with [35S]methionine revealed that PG increased reductase activity by inhibiting HMGR degradation without affecting the synthesis of HMGR. The stimulation of HMGR activity by progesterone could be accounted for by a continuous synthesis of HMGR while its degradation was retarded. In the presence of LDL, the activity of HMGR in IEC-6 cells was effectively inhibited, however PG was able to stimulate HMGR in the presence of LDL. This effect was not due to an interference of normal cellular metabolism of LDL, since PG had no effect on the cellular uptake and lysosomal degradation of 125I-LDL. PG did not affect of the rate of lysosomal hydrolysis of [3H]cholesteryl linoleate-LDL. The free [3H]cholesterol derived from [3H]cholesteryl linoleate-LDL moved to the cell membrane and effluxed to HDL3 in the medium at the same rate in the presence or absence of PG. Although PG did not affect LDL metabolism, pre-treatment of cells with LDL delayed the onset of HMGR stimulation by PG. In IEC-6 cells deprived of LDL for 24 h, the HMGR activity was stimulated immediately following PG addition. In cells pre-treated with LDL for 24 h, the stimulation was delayed by 4 h. Treatment of cells with 25-hydroxycholesterol completely prevented PG stimulation of HMGR activity. We propose that the stimulation of HMGR activity in the presence or absence of LDL is related to the ability of PG to attenuate the formation and/or action of intracellular HMGR repressor molecules which accelerate the degradation of HMGR.

Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevalonate.

We have examined the mechanisms of sterol-independent regulation of the expression of 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase by mevalonate in Chinese hamster ovary (CHO) cells. Serum lipoproteins, 25-hydroxycholesterol, or mevalonate each repress HMG-CoA reductase activity by fivefold or more, and mevalonate lowers the rate of reductase synthesis by twofold. However, while the expression of the HMG-CoA reductase promoter construct, T42 delta CAT, in stable transfectants is also repressed by serum lipoproteins and 25-hydroxycholesterol, mevalonate is without effect. In addition, while 25-hydroxycholesterol reduces the steady-state level of endogenous HMG-CoA reductase mRNA by more than threefold, mevalonate again has no effect. Mevalonate does partially regulate the expression of both the artificial promoter construct pTK-Kx3-CAT, containing three copies of the sterol regulatory element, SRE-1, and the full-length LDL receptor promoter construct, pLDLRCAT-6500 as well as the expression of functional LDL receptors. This transcriptional regulation appears to be mediated by sterol end products generated from added mevalonate. In CHO cells starved for mevalonate due to a mutation in the biosynthetic pathway, addition of 20 mM mevalonate accelerates the rate of degradation of HMG-CoA reductase by threefold whether new sterol biosynthesis is blocked or not. In such cells, addition of 25-hydroxycholesterol, by itself, also decreases the half-life of reductase from 11.6 to 2.3 h. In contrast, in cells acutely treated with a reductase inhibitor, sterol-accelerated degradation of reductase is only observed in the presence of submillimolar level of mevalonate. We conclude that large concentrations of exogenous mevalonate fail to generate a transcriptional regulator of HMG-CoA reductase in CHO cells but do lead to the formation of translational regulator(s) of reductase synthesis. In contrast, sterol regulators derived from exogenous mevalonate appear to be capable of downregulating the LDL receptor promoter. We further conclude that in the absence of pretreatment with a reductase inhibitor, the regulatory signals generated by sterols and nonsterols for accelerated degradation of HMG-CoA reductase are mutually independent. However, the enzyme synthesized in the presence of reductase inhibitors appears to exhibit an obligatory corequirement for low-dose mevalonate for sterol-accelerated degradation.

A functional role for vimentin intermediate filaments in the metabolism of lipoprotein-derived cholesterol in human SW-13 cells.

Numerous studies have indicated that cytoplasmic intermediate filaments (cIFs) can associate with cellular lipids. To determine if these interactions might have functional consequences, we have studied the lipid metabolism of human SW-13 adrenal tumor cell lines that either contain vimentin-type cIFs (vim+) or lack any detectable cIF network (vim-). Although there were no significant differences in phospholipid or glyceride synthesis, vim- cell lines had elevated levels of cholesterol synthesis and decreased cholesterol esterification, compared with vim+ cells. These differences in cholesterol synthesis and esterification were found to be due to an impaired ability of vim- cells to utilize low density lipoprotein (LDL)-derived cholesterol, although receptor-mediated endocytosis of LDL and the capacity of these cells to esterify endogenously produced cholesterol were not affected. Expression of a mouse vimentin cDNA in stably transfected cell lines, derived from vim- cells, restored the capacity of these cells to utilize LDL cholesterol. The uptake and metabolism of [3H]cholesterol linoleate-loaded LDL showed that the impaired ability of vim- cells to esterify LDL cholesterol was not associated with an accumulation of cellular free cholesterol but rather an increase in the appearance of [3H]cholesterol in the culture medium. These studies indicate that in SW-13 cells, the intracellular movement of LDL-derived cholesterol from the lysosome to the site of esterification is a vimentin-dependent process.

Post-transcriptional regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by 24(S),25-oxidolanosterol.

We have examined the mechanism of regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by 24(S),25-oxidolanosterol, a C30-sterol naturally occurring in mammalian tissues. In the absence of enzymatic demethylation to the C27-sterol, 24(S),25-epoxycholesterol, oxidolanosterol is shown to be a post-transcriptional regulator of reductase synthesis in both primary rat hepatocytes and Chinese hamster ovary cells. Under these conditions, oxidolanosterol also increases the rate of degradation of reductase protein in these cells. When demethylation is not inhibited, oxidolanosterol treatment produces transcriptional regulation of sterol-sensitive genes in Chinese hamster ovary cells. In contrast to previous findings with the oxygenated C27-sterol, 25-hydroxycholesterol, oxidolanosterol can act as a post-transcriptional regulator in cells starved for mevalonate. These findings are consistent with the hypothesis that oxidolanosterol down-regulates sterol synthesis in a fashion mechanistically distinct from that of C27-sterols.

Effects of specific inhibition of sterol biosynthesis on the uptake and utilization of low density lipoprotein cholesterol by HepG2 cells.

Treatment of HepG2 cells in lipoprotein-deficient media with 4,4,10 beta-trimethyl-trans-decal-3 beta-ol (TMD) abolished the incorporation of [3H]acetate into cholesterol with concomitant accumulation of squalene 2,3(S)-oxide and squalene 2,3(S):22(S),23-dioxide, indicating a specific inhibition of oxidosqualene cyclase. The activity of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase was affected in a biphasic manner, being inhibited by 30% at low concentrations of TMD and stimulated by 30% at concentrations that completely shut down oxidosqualene cyclase. Treatment with TMD (greater than 20 micrograms/ml) doubled the specific binding and internalization of low density lipoproteins (LDL) and also enhanced their degradation to a degree comparable to that produced by lovastatin, a well-known inhibitor of HMG-CoA reductase. The enhanced binding of LDL to HepG2 cells appeared to occur as a result of an increase in the number of binding sites with no change in their binding affinity for the lipoprotein. At concentrations that completely inhibited cholesterol biosynthesis, TMD did not affect the ability of LDL-derived cholesterol to stimulate cholesterol esterification by seven- to tenfold or to stimulate bile acid secretion to a lesser degree. However, TMD treatment inhibited overall bile acid secretion by 75-85%. The compound had no inhibitory effect on the rates of secretion of either apolipoprotein B or of cholesterol by HepG2 cells into the culture medium. These data demonstrate that a specific inhibition of the sterol branch of isoprenoid biosynthetic pathway in hepatic cells by TMD is sufficient to induce the expression of LDL receptors and that the cholesterol delivered by LDL is available for normal metabolic purposes of the cell.

Defective elongation of fatty acids in a recessive 25-hydroxycholesterol-resistant mutant cell line.

The Chinese hamster ovary recessive mutant, crB, has been selected for its resistance to the cytotoxic effects of 25-hydroxycholesterol in sterol-free media (Sinensky, M., Logel, J., and Torget, R. (1982) J. Cell. Physiol. 113, 314-319). Growth of crB in a chemically defined lipid-poor medium is very slow and is enhanced by a mixture of saturated and unsaturated fatty acids. Incorporation of [3H]acetate into total fatty acids is 4-fold lower in crB compared to that in parental Chinese hamster ovary K1 and in contrast to the wild-type cells, crB cells are unable to synthesize either stearate or oleate. In addition, crB cells can not elongate exogenous palmitate, while they are capable of desaturating exogenous stearate. The mutant cells are also pleiotropically defective in the regulation of mRNA levels for the enzymes of cholesterol biosynthesis. 25-Hydroxycholesterol is a poor regulator of the synthesis and degradation of the rate-limiting enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase in crB in comparison to the wild-type Chinese hamster ovary K1 cells. The defect in the elongation of fatty acids is reversed in revertants of crB selected for their ability to grow in lipid-poor medium. Such revertants exhibit normal regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity by 25-hydroxycholesterol. Regulation of reductase activity in crB cells can also be restored by supplementing the culture medium with a mixture of fatty acids that restores normal growth rate. The defective regulation of reductase in crB does not appear to be due to nonspecific adverse effects of fatty acid starvation nor is it due to any gross change in the fatty acid composition of cellular phospholipids. These results strongly suggest a direct relationship between the fatty acid auxotrophy of crB and defective regulation of the enzymes of cholesterol biosynthesis.

Sterol-independent regulation of 3-hydroxy-3-methylglutaryl-CoA reductase by mevalonate in Chinese hamster ovary cells. Magnitude and specificity.

In this paper, we assess the relative degree of regulation of the rate-limiting enzyme of isoprenoid biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, by sterol and nonsterol products of mevalonate by utilizing cultured Chinese hamster ovary cells blocked in sterol synthesis. We also examine the two other enzymes of mevalonate biosynthesis, acetoacetyl-CoA thiolase and HMG-CoA synthase, for regulation by mevalonate supplements. These studies indicate that in proliferating fibroblasts, treatment with mevalonic acid can produce a suppression of HMG-CoA reductase activity similar to magnitude to that caused by oxygenated sterols. In contrast, HMG-CoA synthase and acetoacetyl-CoA thiolase are only weakly regulated by mevalonate when compared with 25-hydroxycholesterol. Furthermore, neither HMG-CoA synthase nor acetoacetyl-CoA thiolase exhibits the multivalent control response by sterol and mevalonate supplements in the absence of endogenous mevalonate synthesis which is characteristic of nonsterol regulation of HMG-CoA reductase. These observations suggest that nonsterol regulation of HMG-CoA reductase is specific to that enzyme in contrast to the pleiotropic regulation of enzymes of sterol biosynthesis observed with oxygenated sterols. In Chinese hamster ovary cells supplemented with mevalonate at concentrations that are inhibitory to reductase activity, at least 80% of the inhibition appears to be mediated by nonsterol products of mevalonate. In addition, feed-back regulation of HMG-CoA reductase by endogenously synthesized nonsterol isoprenoids in the absence of exogenous sterol or mevalonate supplements also produces a 70% inhibition of the enzyme activity.

Regulation of sterol biosynthesis and of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in cultured cells by progesterone.

Treatment of rat intestinal epithelial cells in culture (IEC-6) with progesterone (10 micrograms/ml) caused a strong inhibition of cholesterol biosynthesis as indicated by a decreased incorporation of radiolabel from [3H]acetate. This inhibition was accompanied by an accumulation of radioactivity in an intermediate which coeluted with authentic desmosterol upon high performance liquid chromatography (HPLC). In addition, treatment of cells with progesterone caused lesser accumulation of radiolabel in products with retention times (RT) of 7.9 and 13.5 min on reverse-phase HPLC. The RT-13.5 compound was tentatively identified as cholesta-5,7,24-trien-3 beta-ol based on its relative retention and on its conversion to cholesterol upon incubation with untreated cells. The RT-7.9 compound was identified as 24 (S),25-epoxycholesterol (S-EC) based on its coelution with authentic S-EC and by its conversion to 25-hydroxycholesterol upon reduction with LiAlH4. Incubation of IEC-6 cells with chemically prepared S-EC resulted in dose-dependent suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity within 6 h (I50 = 0.3 microM). Pretreatment of cells with progesterone prevented this suppressive effect. No suppression of reductase activity was observed in progesterone-treated cells in spite of obvious accumulation of S-EC in amounts sufficient to effect regulation; instead, a 2-3-fold increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity occurred within a 24-h period. Following the removal of progesterone from the culture medium, reductase activity declined rapidly over the next 6 h. However, IEC-6 cells could not metabolize S-EC, derived either endogenously or exogenously, during a similar time frame; nor did progesterone affect the uptake of exogenous S-EC by IEC-6 cells. These results show that although progesterone treatment of cultured cells promotes the synthesis of a natural oxysterol suppressor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the continued presence of progesterone prevents the regulatory action of S-EC. The unique nature of this interference is high-lighted by the observation that progesterone could not prevent the suppression of reductase activity by either 25-hydroxycholesterol or mevalonolactone.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis by oxylanosterols.

Treatment of rat intestinal epithelial cell cultures with the oxidosqualene cyclase inhibitor, 3 beta-[2-(diethylamino)-ethoxy]androst-5-en-17-one (U18666A), resulted in an accumulation of squalene 2,3:22,23-dioxide (SDO). When U18666A was withdrawn and the cells were treated with the sterol 14 alpha-demethylase inhibitor, ketoconazole, SDO was metabolized to a product identified as 24(S),25-epoxylanosterol. To test the biological effects and cellular metabolism of this compound, we prepared 24(RS),25-epoxylanosterol by chemical synthesis. The epimeric mixture of 24,25-epoxylanosterols could be resolved by high performance liquid chromatography on a wide-pore, non-endcapped, reverse phase column. Both epimers were effective suppressors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity of IEC-6 cells. The suppressive action of the natural epimer, 24(S),25-epoxylanosterol, but not that of 24(R),25-epoxylanosterol could be completely prevented by ketoconazole. IEC-6 cells could efficiently metabolize biosynthetic 24(S),25-epoxy[3H]anosterol mainly to the known reductase-suppressor 24(S),25-epoxycholesterol. This metabolism was substantially reduced by ketoconazole. These data support the conclusion that 24(S),25-epoxylanosterol per se is not a suppressor of HMG-CoA reductase activity but is a precursor to a regulatory oxysterol(s). It has recently been reported that 25-hydroxycholesterol can occur naturally in cultured cells in amounts sufficient to effect regulation of HMG-CoA reductase (Saucier et al. 1985. J. Biol. Chem. 260: 14571-14579). In order to investigate the biological effects of possible precursors of 25-hydroxycholesterol, we chemically synthesized 25-hydroxylanosterol and 25-hydroxylanostene-3-one. Both oxylanosterol derivatives suppressed cellular sterol synthesis at the level of HMG-CoA reductase. U18666A had the unusual effect of potentiating the inhibitory effect of 25-hydroxylanostene-3-one but did not influence the effect of other oxylanosterols. All the oxylanosterols, with the exception of 25-hydroxylanostene-3-one, enhanced intracellular esterification of cholesterol. The foregoing observations support consideration of oxylanosterols as playing an important role in the biological formation of regulatory oxysterols that modulate sterol biosynthesis at the level of HMG-CoA reductase.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by oxysterol by-products of cholesterol biosynthesis. Possible mediators of low density lipoprotein action.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34, reductase) activity was studied in cultured rat intestinal epithelial cells using 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one ( U18666A ), an inhibitor of 2,3- oxidosqualene cyclase (EC 5.4.99.7, cyclase) that causes cellular accumulation of squalene 2,3:22,23-dioxide ( Sexton , R. C., Panini , S.R., Azran , F., and Rudney , H. (1983) Biochemistry 22, 5687-5692). Treatment of cells with U18666A (5-50 ng/ml) caused a progressive inhibition of reductase activity. Further increases in the level of the drug paradoxically lessened the inhibition such that at a level of 1 microgram/ml, no inhibition of enzyme activity was observed. Cellular metabolism of squalene 2,3:22,23-dioxide to compounds with the chromatographic properties of polar sterols led to an inhibition of reductase activity that could be prevented by U18666A (1 microgram/ml). The drug was unable to prevent the inhibition of enzyme activity by 25-hydroxycholesterol or mevalonolactone, but totally abolished the inhibitory action of low density lipoproteins. Pretreatment with U18666A did not affect the ability of cells to degrade either the apoprotein or the cholesteryl ester component of low density lipoproteins. These results suggest that oxysterols derived from squalene 2,3:22,23-dioxide may act as physiological regulators of reductase and raise the possibility that the suppressive action of low density lipoproteins on reductase may be partially or wholly mediated by such endogenous oxysterols generated through incomplete inhibition of the cyclase.

Effects of 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one on the synthesis of cholesterol and ubiquinone in rat intestinal epithelial cell cultures.

The relationship between cholesterol and ubiquinone synthesis in rat intestinal epithelial cell cultures was examined by using 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one hydrochloride (U18666A). Addition of U18666A to cells caused a greater than 90% inhibition of incorporation of [3H]acetate into cholesterol and an apparent large increase in the incorporation of [3H]acetate and [3H]mevalonate into ubiquinone. However, the incorporation of 4-hydroxy[U-14C]benzoate, a ring precursor of ubiquinone, was unchanged. The apparent increase of 3H incorporation into ubiquinone was found to be due to the formation of a contaminant that has been identified as squalene 2,3:22,23-dioxide. Following incubation of cells with U18666A, its removal from the medium resulted in a decrease in squalene 2,3:22,23-dioxide labeling and a corresponding increase in the polar sterol fraction. These results demonstrate that U18666A inhibits the reaction catalyzed by 2,3-oxidosqualene cyclase (EC 5.4.99.7). As a result, the isoprenoid precursors are diverted not to ubiquinone as has been suggested but to squalene 2,3:22,23-dioxide, a metabolite not on the cholesterol biosynthetic pathway. Removal of the drug allows cyclization of squalene 2,3:22,23-dioxide, leading to formation of compounds with chromatographic properties of polar sterols.

Short term reversible modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in isolated epithelial cells from rat ileum. Regulation of phosphorylation dephosphorylation by bicarbonate.

In ileal epithelial cells isolated through incubation of gut loops in phosphate-buffered saline containing EDTA, 3-hydroxy-3-methylglutaryl coenzyme A reductase was found to exist prdominantly in an active (dephosphorylated) form. However, in cells obtained by scraping the intestine, the enzyme was mostly inactive (phosphorylated) and could be activated several-fold on incubation with a crude phosphatase preparation. Reductase activity in microsomes from ileal epithelial cells could be inactivated extensively by incubation with Mg . ATP in the absence of any exogenous factors. When cells isolated in phosphate-buffered saline were incubated in bicarbonate-containing buffers, there was a time-dependent decrease in the apparent activity of 3-hydroxy-3-methylglutaryl CoA reductase with little change in the total activity of the enzyme. On transferring the cells to a buffer without bicarbonate, the decrease in the apparent activity of the enzyme was fully reversed. There was no change in both the apparent and the total activities of reductase when cells were incubated in buffers lacking bicarbonate. Sterol synthetic rates in isolated intestinal cells were reflective of the fraction of reductase in the active form. These results strongly suggest the operation of a phosphorylation-dephosphorylation mechanism for short term modulation of 3-hydroxy-3-methylglutaryl CoA reductase activity in the intact mucosal cell. The bicarbonate anion appears to function as an activator of this modulation system.

Distribution of 3-hydroxy-3-methylglutaryl coenzyme A reductase and alkaline phosphatase activities in isolated ileal epithelial cells of fed, fasted, cholestyramine-fed, and 4-aminopyrazolo[3,4-d]pyrimidine-treated rats.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, E.C. 1.1.1.34), the major rate-limiting enzyme of the sterol biosynthetic pathway, was studied in ileal epithelial cells isolated in a villus-to-crypt gradient according to Weiser (Weiser, M. M. 1973. J. Biol. Chem, 248:2536-2541). Alkaline phosphatase (E.C. 3.1.3.1) served as a marker for the mature villus cells. Protease effects on activity determinations were negligible. The intracellular location of HMG-CoA reductase could not be precisely determined. The activity of ileal reductase was predominantly associated with the less differentiated lower villus and crypt cells, while the reverse gradient occurred with alkaline phosphatase. This distribution of enzymes persisted in both fed and fasted rats injected with control saline-phosphate, although fasting decreased total reductase units in the ileum by 86% in 72 hr. Treatment with cholestyramine and with 4-aminopyrazolo[3,4-d]pyrimidine (APP) enhanced reductase activity in ileal cells. The percent stimulation in both cases was higher in the upper villus cells than in the crypt cells, leading to abolition of the gradient in enzyme activity. However, APP treatment caused a 98% loss in total alkaline phosphatase units and a 55% loss in total epithelial cell protein in 72 hr. Thus, there was no increase in total reductase units. These data show that APP affects ileal cell metabolism directly. Furthermore, it appears that the regulation of sterol synthesis in the intestinal mucosa, via HMG-CoA reductase, involves a complex interplay of the effects exerted by the level of alimentation, the enterohepatic circulation of bile, and the levels of plasma lipoproteins.