Effects of porcine relaxins upon uterine hypertrophy and protein metabolism in mice

Two variant forms of porcine relaxin (B and C) are active in producing relaxation of the guinea pig pubic symphysis and in effecting uterine growth in rats. Only relaxin B, however, is active in the mouse pubic ligament assay. These two hormones were compared in mice for their effects upon uterine growth and incorporation of radioactively labeled proline into soluble protein and collagen in vitro and in vivo. Both relaxin B and relaxin C produced an early (3-hr) elevation in in vitro protein synthesis and a later (6-hr) increase in collagen incorporation of proline at the time when the uterotrophic effect was maximal. In vivo effects of relaxin C on the uterus were in some cases greater than relaxin B in contrast to the complete inactivity of the former upon the pubic ligament of the mouse. These findings suggest a high degree of tissue specificity for relaxin stimulation, a variability in responsiveness among tissues in the same animal, and perhaps a primary role of relaxin in uterine function with pelvic relaxation representing a secondary function which has developed in certain species.     

A sensitive method for the separation and quantitation of (14C)proline and (14C)hydroxyproline from the collagen of rat uterus

A specific and sensitive method is described for the isolation and quantitation of [¹⁴C]proline and [¹⁴C]hydroxyproline from uterine collagen of the immature rat. Selectivity is achieved in this isolation by using a protease-free bacterial collagenase. There is complete release of hydroxyproline from uterine protein if the latter is suspended by sonication prior to treatment with collagenase. There is a consistent recovery of [¹⁴C]proline and [¹⁴C]hydroxyproline when they are added to protein hydrolysates of uterus and then subjected to the procedures required for their isolation and quantitation. It is possible using this method to determine the incorporation of [¹⁴C]proline into collagen of the rat uterus and to quantitate its conversion to [¹⁴C]hydroxyproline. Coupled with the colorimetric methods for proline and hydroxyproline, it is also possible to determine their specific activity.     

Proline and hepatic lipogenesis

The effects of proline on lipogenesis in isolated rat hepatocytes were determined and compared with those of lactate, an established lipogenic precursor. Proline or lactate plus pyruvate increased lipogenesis (measured with 3H2O) in hepatocytes from fed rats depleted of glycogen in vitro and in hepatocytes from starved rats. Lactate plus pyruvate but not proline increased lipogenesis in hepatocytes from starved rats. ( - )-Hydroxycitrate, an inhibitor of ATP-citrate lyase, partially inhibited incorporation into saponifiable fatty acid of 3H from 3H2O and 14C from [U-14C]lactate with hepatocytes from fed rats. Incorporation of 14C from [U-14C]proline was completely inhibited. Similar complete inhibition of incorporation of 14C from [U-14C]proline by ( - )-hydroxycitrate was observed with glycogen-depleted hepatocytes or hepatocytes from starved rats. Inhibition of phosphoenolpyruvate carboxykinase by 3-mercaptopicolinate did not inhibit the incorporation into saponifiable fatty acid of 3H from 3H2O or 14C from [U-14C]proline or [U-14C]lactate. Both 3-mercaptopicolinate and ( - )-hydroxycitrate increased lipogenesis (measured with 3H2O) in the absence or presence of lactate or proline with hepatocytes from starved rats. The results are discussed with reference to the roles of phosphoenolpyruvate carboxykinase, mitochondrial citrate efflux, ATP-citrate lyase and acetyl-CoA carboxylase in proline- or lactate-stimulated lipogenesis.     

Effect of α,α'-dipyridyl on Exogut Formation in Vegetalized Embryos of the Sea Urchin

In presumptive vegetalized embryos, obtained by 3-hr treatment with chloramphenicol at the 16–32 cell stage, the rates of [¹⁴C]proline incorporation into the collagen fraction and production of the [¹⁴C]hydroxyproline residues increased during development between 16 hr (equivalent to mesenchyme blastula stage) and 40 hr (the early pluteus stage) after fertilization at 20°C. In presumptive vegetalized embryos, the radioactivity of [¹⁴C]hydroxyproline residues was higher at the mesenchyme blastula stage (16 hr after fertilization), but lower at the post-gastrula stage than in normal embryos. In normal embryos at the post-gastrula stage, [¹⁴C]hydroxyproline residues were mainly found in isolated spicules, and the amounts of [¹⁴C]hydroxyproline residues in other parts were much lower than in vegetalized embryos, which had few, if any, spicules. α, α'-Dipyridyl, an inhibitor of prolyl hydroxylase, inhibited the hydroxylation of [¹⁴C]proline residues in presumptive vegetalized and normal embryos, and blocked the formation of the archenteron and exogut.     

Effect of L-3,4-dehydroproline on collagen synthesis and prolyl hydroxylase activity in mammalian cell cultures.

The incorporation of DL-3,4-dehydro[14C]proline into collagen and total protein of 3T3 cells occurred at approximately one-fifth the rate observed for L-[14C]proline. Addition of L-3,4-dehydroproline to the culture medium inhibited markedly the incorporation of [14C]glycine and L-[3H]lysine into the collagen of 3T3 cells, but there was only slight inhibition of the incorporation of the radiolabeled amino acids into total cellular proteins, indicating that the action of L-3,4-dehydroproline is specific for collagen. When 1 mM L-3,4-dehydroproline was added to the culture medium the [14C]hydroxyproline content was reduced 40% in the cell layer and 70% in the medium. The D isomer of 3,4-dehydroproline did not inhibit [14C]hydroxyproline formation. These findings indicate that L-3,4-dehydroline reduced the hydroxylation of the susceptible prolyl residues in the collagen molecule and the secretion of collagen from the cell. The reduction in the hydroxyproline content is probably related in part to a reduction in the activity of prolyl hydroxylase; when various mammalian cell cultures were exposed to 0.2 mM L-3,4-dehydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroxylase was not affected. Under these conditions, cell growth and lactic dehydrogenase required protein synthesis. Removal of L-3,4-dehydroproline from the growth medium resulted in a time-dependent increase in the specific activity of prolyl hydroxylase.     

Progesterone inhibits the uterotrophic effect of relaxin in immature rats

Injection of progesterone for 3 days before treatment with relaxin inhibited the trophic effect of the peptide in both estrogen-primed and unprimed uteri. The depression in collagen concentration and increase in apparent rate of proline incorporation into collagen induced by relaxin alone were also eliminated, indicating a fundamental blockade of the effect of relaxin in this experimental design as well as a close association of changes in collagen concentration with tissue hypertrophy. The effect of relaxin on incorporation of proline into soluble protein was not blocked by progesterone, however, suggesting a separate mechanism for this anabolic effect of relaxin.     

The embryonic rat parietal yolk sac. The role of the parietal endoderm in the biosynthesis of basement membrane collagen and glycoprotein in vitro.

Basement membrane biosynthesis in vitro was studied in a rapidly growing embryonic tissue, the rat parietal yolk sac. This tissue consists of a thick, nonvascular basement membrane (Reichert's membrane) separating two cellular layers (parietal endoderm and trophoblast). Morphologically, Reichert's membrane appeared similar to other basement membranes. Previous analysis of the amino acid and carbohydrate composition of acellular Reichert's membrane showed it to be typical of basement membranes isolated from other tissues and species. Analysis of [14-C]proline incorporation and hydroxy [14-C]proline synthesis during the third quarter ogestation in vitro showed that basement membrane collagen synthesis in the parietal yolk sac was maximal around the 14th day of gestation. At this time, basement membrane collagen represented nearly 10% of the newly synthesized protein. The collagen synthesized in this system was characteristic of basement membrane collagen in that about 11% of the total hydroxy [14-C]proline was present as the 3-isomer. In addition, after incubation in the presence of [14-C]lysine, 83 to 94% of the hydroxy[14-C]lysine was glycosylated, with the predominant form being glucosylgalactosylhydroxy[14-C]lysine. When the parietal endoderm and trophoblast were incubated separately with [14-C]proline, it was determined that the former was solely responsible for the synthesis of basement membrane collagen since essentially all of the 4-hydroxy[14-C]proline was associated with this cell type. Autoradiographic experiments with [3-H]glucosamine also served to localize the synthesis of noncollagen basement membrane glycoprotein components to the parietal endoderm. As with the results reported for basement membrane collagen secretion in embryonic chick lens cells, there appeared to be approximately a 60-min delay between the incorporation of [14-C]proline into protein and the secretion of collagen as measured by the appearance of 4-hydroxy[14-C]proline in the culture medium. Experiments utilizing [3H]glucosamine to monitor glycoprotein synthesis did not show a delay between the incorporation of [3H]glucosamine and the secretion of nondialyzable 3-H into the medium. The results obtained using the parietal yolk sac system to study basement membrane biosynthesis were compared to those previously obtained using the kidney glomerular and embryonic chick lens systems. It was concluded that the parietal yolk sac system is superior for a number of reasons: (a) the extracellular matrix appeared to contain only basement membrane components; there was no contamination by acid mucopolysaccharides or other types of collagen; (b) only a single cell type appeared to be responsible for the synthesis of basement membrane components; and (c) a relatively large percentage of the newly synthesized protein was basement membrane collagen.     

Extracellular matrix metabolism by chondrocytes. VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[3H]proline into hydroxyproline and [3H]acetate into glycosaminoglycans, was shown to be depressed by 58% and 39%, respectively, by the addition of exogenous proteoglycan at a concentration of 10 mg/ml growth media. The incorporation of L-[3H]proline into acid-insoluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity on the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-translational modification of collagen and proteoglycan.     

The in vivo inhibition of collagen synthesis and the reduction of prolyl hydroxylase activity by 3,4-dehydroproline.

Various proline analogs and iron chelators were tested for their effect on collagen formation which occurs in the uterus of the immature rat following the administration of estradiol-17β. dl-3,4-Dehydroproline, l-α-azetidine-2-carboxylic acid and l-pyroglutamic acid reduced the estradiol-17β stimulated formation of hydroxyproline which occurs in the uterus following administration of the hormone while l-thiazolidine-4-carboxylic acid was without effect on this response. The activity of the d- and l-isomers of 3,4-dehydroproline was compared with the racemic mixture; the l-isomer was twice as active as the latter, while the d-isomer was only half as active. l-3,4-Dehydroproline was approximately four times as potent as l-α-azetidine-2-carboxylic acid, the second most active analog of those tested. dl-3,4-Dehydroproline inhibited the incorporation of l-[¹⁴C]proline into the proline and hydroxyproline of uterine collagen; it also inhibited the incorporation of [¹⁴C]glycine into collagen while having less effect on the incorporation of these amino acids into noncollagen protein. These results indicate dl-3,4-dehydroproline is a fairly specific and potent inhibitor of collagen formation in vivo.These observations indicate that dl-3,4-dehydroproline reduces the hydroxylation of prolyl residues in collagen. Presumably, this occurs in part due to the incorporation of the analog into the collagen molecule in place of proline. It is probably also related to a reduction of prolyl hydroxylase activity which can be demonstrated in the tissues of animals treated with 3,4-dehydroproline. A significant reduction of prolyl hydroxylase activity was shown to persist in the uterus, lung, and heart for approximately 24 h following a single intraperitoneal dose of dl-3,4-dehydroproline (200 mg/kg).     

The biosynthesis of basement-membrane collagen by isolated rat glomeruli.

A technique is described for the rapid isolation of highly purified preparations of viable glomeruli from rat kidney cortex. The synthesis of protein as judged by the incorporation of [14C]proline into non-diffusible material was shown to be linear for up to 6 h. The synthesis of collagen, measured as non-diffusible 4-hydroxy[14C]proline, was also linear over this period but represented only a small proportion of total protein synthesis. Similar studies conducted in vivo confirmed that collagen synthesis accounted for less than 5% of total protein synthesis in glomeruli. When isolated glomeruli were incubated with [14C]proline, it was found that approximately 16% of the hydroxyproline present in the collagenous component occurred as the 3-isomer. When glomeruli were incubated with [14C]lysine over 90% of the hydroxy[14C]lysine synthesised was glycosylated and most of the glycosylated hydroxy[14C]lysine was present as glucosyl-galactosyl-hydroxy[14C]lysine. The size of the basement membrane collagen synthesised by the isolated glomeruli was estimated by treating the 14C-labelled protein with mercaptoethanol and sodium dodecyl sulphate and then chromatographing the 14C-labelled protein on an agarose column equilibrated and eluted with buffer containing 0.1% (w/v) sodium dodecyl sulphate. The initial form of [14C]collagen synthesised was found to consist of polypeptide chains which had molecular weights of approximately 140 000 and which were shown to be distinctly larger than the polypeptide chains from embryonic chick tendon procollagen. Also when glomeruli were labelled with [14C]proline for 2 h and chased with unlabelled proline for 4 h there was a time-dependent conversion of the initially synthesised collagen moiety to collagen polypeptide chains which co-chromatograph with tendon pro-alpha chains (molecular weight approx. 120 000).     

Inhibition of lipogenesis by vasopressin and angiotensin II in glycogen-depleted hepatocytes

Vasopressin and angiotensin II inhibited lipogenesis (measured with 3H2O) in hepatocytes from fed rats. Inhibition was also observed with hepatocytes from fed rats which had been depleted of glycogen in vitro and incubated with lactate + pyruvate (5 mM + 0.5 mM) as substrates. The inhibitory actions of the hormones are therefore independent of hormone-mediated changes in glycogenolytic or glycolytic flux from glycogen, and thus the site(s) of hormone action must be subsequent to the formation of lactate. (-)Hydroxycitrate, a specific inhibitor of ATP-citrate lyase, decreased lipogenesis in hepatocytes from fed rats incubated with lactate + pyruvate by approx. 51% but had little effect on lipogenesis in glycogen-depleted hepatocytes similarly incubated. There was parallel inhibition of incorporation of 14C from [U-14C]lactate into fatty acid and lipogenesis as measured with 3H2O in each case. Thus depletion of glycogen, or conceivably the process of glycogen-depletion (incubation with dibutyryl cyclic AMP) causes a change in the rate-determining step(s) for lipogenesis from lactate. Vasopressin and angiotensin II also decreased lipogenesis and incorporation of 14C into fatty acids in glycogen-depleted hepatocytes provided with [U-14C]proline as opposed to [U-14C]-lactate. However, proline-stimulated lipogenesis was inhibited by (-)hydroxycitrate, and proline-stimulated lipogenesis and incorporation of 14C from [U-14C]-proline were not decreased in parallel by this inhibitor (inhibition of 52% and 85% respectively). It is inferred that lactate and proline stimulate lipogenesis by different mechanisms and incorporation of 14C from [U-14C]proline and [U-14C]lactate into fatty acid occurs via different routes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative study of tissue collagen metabolism

A procedure for the quantification of various parameters of metabolism of collagen in fibrotic mouse liver has been developed. The method involves derivatization of hydroxyproline, a marker of collagen, with dansyl chloride, high-performance liquid chromatography of the derivative on an octadecyl C-18 column, and its detection by fluorescence. This assay improves upon existing procedures in several respects: It extends the analysis so that not only the collagen content of the tissue but also the metabolism of collagen is determined at levels found intracellularly. It is sensitive enough to quantify 0.1-10 nmol of hydroxyproline, and it includes three major amino acids (hydroxyproline, glycine, and proline) of collagen and two assay controls; it generates information on both the purity and quantity of collagen in each assay. The determination of specific activity of intracellular free [14C]proline, which is the precursor of protein-bound hydroxyproline, defines the specific activity of [14C]hydroxyproline of collagen converted from precursor residues of [14C]proline by the action of prolyl hydroxylase. The specific activity of [14C]hydroxyproline can be used for the evaluation of collagen synthesis and secretion and intracellular and extracellular degradation of the newly synthesized and secreted collagen in the tissue. The determination of specific activities of [14C]hydroxyproline and [14C]proline and of the ratio of [14C]hydroxyproline to [14C]proline of newly secreted collagen provides information concerning the extent of hydroxylation of [14C]proline residues of newly synthesized collagen.     

Extracellular matrix metabolism by chondrocytes: VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[³H]proline into hydroxyproline and [³H]acetate into glycosaminoglycans, was shown to be depressed by 59% and 39%, respectively, by the addition of exogenous proteoglycan at a concetration of 10 mg/ml growth media. The incorporation of L-[³H]proline into acid-in-soluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity of the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-traslational modification of collagen and proteoglycan.     

Biosynthesis of basement membrane collagen by rabbit corneal endothelium in vitro.

The synthesis of collagen has been demonstrated in endothelial cells of Descemet's membrane isolated from rabbit cornea. Incorporation of [14C]proline and [14C]lysine into nondialyzable protein was measured in the medium and cell fraction after incubating Descemet's membrane for up to 5 hours. In the [14C]collagen synthesized by the endothelium, 15% of the hydroxy[14C]proline was present as the 3-isomer. About 98% of the hydroxy[14C]lysine in the 14C-labeled-protein found in the medium was glycosylated; 95% of the glycosylated hydroxy[14C]lysine was in the form of the disaccharide glucosyl-galactosyl-hydroxy[14C]lysine. Time course experiments with [14C]proline indicated that there was a delay of about 60 min before significant amounts of [14C]collagen were secreted into the medium. The initial polypeptides of [14C]collagen synthesized by the corneal endothelium had an apparent molecular weight of 155,000. The chemical and physical properties of the [14C]collagen synthesized by rabbit corneal endothelium are consistent with those of basement membrane collagen synthesized by other cell types.     

The consequences of the isotope effect on proline dehydrogenation rates estimated by the tritium loss method.

Loss of tritium from a substrate is often used to estimate the rate of dehydrogenation. However, loss of 3H may be much slower than loss of H because of the tritium isotope effect. In order to assess the impact of the tritium isotope effect, loss of 3H from the C-5 position of proline during dehydrogenation by rat liver mitochondria and bacteroids from soybean (Glycine max [L.] Merrill) nodules was compared with appearance of 14C in products of [14C]proline dehydrogenation. Incubations were carried out in the presence of o-aminobenzaldehyde (added to trap the initial product, delta 1-pyroline-5-carboxylate). The fraction of total 14C products trapped by o-aminobenzaldehyde varied from 0.07 to 0.75 depending upon experimental conditions. With rat liver mitochondria, dehydrogenation of [14C]proline was between 3.27 and 9.25 times faster than dehydrogenation of 3H proline, depending upon assay conditions. Soybean nodule bacteriods dehydrogenated [14C]proline about 5 times faster than [3H]proline. We conclude the following: (i) the rate of proline dehydrogenation may be greatly underestimated by the tritium assay because of the tritium isotope effect, and (ii) the 14C assay may underestimate the rate of proline dehydrogenation if it is assumed that o-aminobenzaldehyde quantitatively traps delta 1-pyrroline-5-carboxylate under all conditions. The simplicity of the tritium assay makes it attractive for routine use. However, its use requires determination of the tritium isotope effect, under the specific conditions of the assay, in order to correct the results. The considerations discussed here have broad applicability to any dehydrogenase assay employing tritium loss.     

Effects of prostaglandin E1 on collagen production and degradation in human fetal lung fibroblasts

We examined the effects of prostaglandin E1 on the production and degradation of collagen in human fetal lung fibroblasts. Percentage collagen production was determined by incubating confluent cultures for 6 h with [3H]proline and either [14C]glycine or [14C]leucine and measuring the relative amounts of radioactivity incorporated into collagenase-sensitive and collagenase-insensitive material. Percentage collagen degradation was determined by measuring hydroxy[14C]proline in a low-molecular-weight fraction relative to total hydroxy[14C]proline. Prostaglandin E1, when present at a concentration as low as 0.25 micrograms/ml, reduced net collagen production by a factor of one-half, from 8 +/- 2 to 4 +/- 1% (P less than 0.05). In contrast, the change in percentage degradation was relatively gradual, rising steadily from the control value of 15 +/- 2 to 33 +/- 2% at 4 micrograms/ml (P less than 0.05). The increase in degradation, while significant, could not account for the total decrease in collagen production. We conclude that prostaglandin E1 exerts its inhibitory effect on collagen production in two essentially independent ways: lowering the rate of synthesis and increasing intracellular degradation. However, the decrease in synthesis is greater than the increase in degradation.     

Tunicamycin inhibits protein glycosylation in suspension cultured soybean cells

Soybean cells in suspension culture incorporate [³H]mannose into dolichyl-phosphoryl-mannose and into lipid-linked oligosaccharides as well as into extracellular and cell wall macromolecules. Tunicamycin completely inhibited the formation of lipid-linked oligosaccharides at a concentration of 5 to 10 micrograms per milliliter, but it had no effect on the formation of dolichyl-phosphoryl-mannose. Tunicamycin did inhibit the incorporation of [³H]mannose into cell wall components and extracellular macromolecules, but even at 20 micrograms per milliliter of antibiotic there was still about 30% incorporation of mannose. The radioactivity in these macromolecules was localized in mannose (70%), rhamnose (20%), galactose (8%), and fucose (2%) in the absence of antibiotic. But when tunicamycin was added, very little radioactive mannose was found in cell wall or extracellular components. The incorporation of [³H]leucine into membrane components and [¹⁴C]proline into cell wall components by these suspension cultures was unaffected by tunicamycin. However, tunicamycin did inhibit the appearance of leucine-labeled extracellular macromolecules, probably because it prevented their secretion.     

Inhibition of collagen synthesis by α, α′-dipyridyl in human skin fibroblasts in culture

Summary In human diploid skin fibroblasts in culture we have shown that nonhydroxylated collagen precursors remain in the cell when proline hydroxylation is inhibited by α, α′-dipyridyl, a chelator of ferrous ions. The inhibition of proline hydroxylation is reversed by addition of fresh medium containing 50 μg per ml of sodium ascorbate, whereupon nonhydroxylated collagen precursors are hydroxylated within the cell and extruded into the medium. Extrusion of collagen already formed within the cell is not appreciably affected by α, α′-dipyridyl inhibition. Under normal conditions collagen is released from the monolayer into the medium within 3 hr of a pulse ofL[¹⁴C]proline. In the presence of α, α′-dipyridyl, about 35% of theL[¹⁴C]proline incorporated into protein is released into the medium within 8 hr as a proline-rich, hydroxyproline-deficient protein; at the same time, approximately 15% of the protein-boundl-[¹⁴C]proline remains in the cell for as long as 12 hr. When proline hydroxylation is restored after 2 and 12 hr of α, α′-dipyridyl inhibition, approximately the same amount of hydroxyproline is formed after each time interval in the monolayer. Therefore, nonhydroxylated collagen precursors retained in the cell are not appreciably degraded during at least 12 hr of inhibition by α, α′-dipyridyl and are extruded into the medium only upon restoration of hydroxylation. This work was supported in part by a grant from the Easter Seal Research Foundation, and by Project 236, Health Services and Mental Health Administration, Department of Health, Education and Welfare, Grant HD-03110 from the National Institute of Child Health and Human Development, an American Cancer Society Institutional Grant (1N 15-J), a General Research Support Award (5-S01-FR-05406) from the National Institutes of Health, a University Research Council Grant, a National Science Foundation Equipment Grant (GB-4577), and a Research Career Development Award (5-K3-AM-5058) from the National Institute of Arthritis and Metabolic Disease (G.K.S.).     

Brain cholesterol XVIII: EFFECt of methylphenidate (Ritalin) on [U-14C] glucose and [2-3H] acetate incorporation.

The effect of a single injection of methylphenidate (Ritalin, 4 mg/kg) on precursor ([2-3H]acetate and [U-14C]glucose) incorporation into brain cholesterol was studied. The drug caused a steady decrease in the concentration of brain cholesterol during the 24-hr period examined. Incorporation studies during this time with [U-14C]glucose indicated higher than normal incorporation for all time periods studied. The most significant incorporation increases took place 2 and 4 hr after drug injection. Experiments using [2-3H]acetate as the sterol precursor gave incorporation values which tended (not significantly) to be lower than control values at 2 and 4 h. The values after 12 hr were less than normal, while the 24-hr group indicated an increase to or slightly higher than normal values. These data suggest that the pharmacological effect of methylphenidate may be due to lowering of brain cholesterol levels directly or on some more basic metabolic process leading to a decreased level of membrane sterols.