Synthesis of ent-25-hydroxycholesterol

25-Hydroxycholesterol (25-HC) appears to play a role in several important biological processes, including regulating cellular cholesterol levels and promoting apoptosis. However, in most cases the mechanisms by which 25-HC elicits its biological effects are not known. Insights into mechanisms of 25-HC action can be gained by studying the activity of its enantiomer (ent-25-HC). ent-25-HC is physically and chemically identical to 25-HC; however, 25-HC and ent-25-HC can be distinguished in chiral environments, like a protein binding site. In order to probe the mechanisms of 25-HC action, we have synthesized the enantiomer of 25-HC (ent-25-HC).     

The effect of low density lipoproteins, cholesterol, and 25-hydroxycholesterol on apolipoprotein B gene expression in HepG2 cells.

The purpose of the present study was to examine the effects of exogenous cholesterol on the apolipoprotein (Apo) B gene expression in HepG2 cells. Pure cholesterol had no significant effect on either the cellular content of cholesteryl esters or the net accumulation of neutral lipids and ApoB in the culture medium. By contrast, addition of 25-hydroxycholesterol increased the net accumulation of cholesteryl esters in cells and medium by 2-3-fold and decreased that of unesterified cholesterol by 50% in both compartments. A 33% reduction in the cellular content of triglycerides was commensurate with a 40% increase in their accumulation in the medium. A significant 3-fold increase in the net accumulation of ApoB in the medium was predominantly due to enhanced secretion of newly synthesized ApoB as established by pulse-chase studies. The stimulation in ApoB secretion was accompanied by a 55% increase in cellular ApoB mRNA. Under these experimental conditions, the low density lipoprotein receptor activity was decreased by only 12-20%. Addition of progesterone prevented the effects of 25-hydroxycholesterol. The changes in the concentration of neutral lipids and ApoB were reflected in the composition of secreted "low-density" lipoproteins. These particles had increased percentage contents of cholesteryl esters and ApoB and a decreased percentage content of unesterified cholesterol in comparison with lipoproteins produced by control cells. The rate of ApoB production was not correlated with the triglyceride mass in the cells but was positively correlated with the cellular and secreted cholesteryl esters and secreted triglycerides. With the exception of unchanged cellular unesterified cholesterol and ApoB mRNA levels, plasma low density lipoprotein had similar, although less pronounced, effects on the production of neutral lipids and ApoB. These results demonstrate that in HepG2 cells the synthesis and secretion of ApoB and cholesteryl esters are tightly coupled and that 25-hydroxycholesterol increased the concentration of ApoB-containing lipoproteins primarily by stimulating their production rather than reducing their catabolism.     

The effect of intracellular calcium oscillations on fluid secretion in airway epithelium

Airway epithelium has been shown to elicit fluid secretion after a rise in intracellular calcium. This rise in intracellular calcium has been shown to display complex oscillations in many species after the binding of particular agonists to extracellular receptors.Fluid secreted by the airway epithelium is used to maintain the depth of the periciliary liquid (PCL) above the apical membrane of the epithelial cells lining the bronchial airways. Previous mathematical models have been published which separately consider the electrophysiology involved in regulating periciliary liquid depth, and the transmission of intracellular calcium waves in airway epithelial tissue. In this paper we present a mathematical model that combines these previous models and allows the effect of oscillations in intracellular calcium on fluid secretion by airway epithelial cells to be investigated. We show that an oscillatory calcium response produces different fluid secretion properties to that elicited by a tonic rise in intracellular calcium. These differences are shown to be due to saturation of the Ca²⁺ activated ion channels.     

Diminution of L cell microvilli following exposure to 25-hydroxycholesterol.

The effect of sterol depletion on the topology of mouse L cells was studied by scanning electron microscopy. Treatment of cells with 25-hydroxycholesterol inhibited the synthesis of cellular sterol and diminished the number of microvilli on the cell surface. Simultaneous addition of mevalonic acid or cholesterol to cells during treatment with the inhibitor prevented the loss of microvilli. These results demonstrate that cholesterol is important in maintaining the ultrastructure of the surface membrane of nucleated mammalian cells.     

The effect of calcium pump inhibitors on the response of intracellular calcium to caffeine in snail neurones

We have measured intracellular free calcium ([Ca(2+)]i) using Fura-2 or Ca(2+)-sensitive microelectrodes in voltage-clamped neurones of the snail, Helix aspersa. Caffeine-induced transient increases in [Ca(2+)]i were normally followed by a brief fall of [Ca(2+)]i below its pre-caffeine level. We investigated the cause of this undershoot by raising [Ca(2+)]i; and by inhibiting the plasma membrane or endoplasmic reticulum Ca ATPases (PMCA or SERCA respectively). When the cell membrane potential was decreased from -60 to -25mV, steady-state [Ca(2+)]i increased. The caffeine-induced transients were smaller while the undershoots were larger than in control conditions. When the PMCA was inhibited by high pH the steady-state [Ca(2+)]i increased by 100-400nM. The caffeine-induced [Ca(2+)]i increase and the subsequent undershoot both became larger. Injection of orthovanadate, which inhibits the PMCA and increases [Ca(2+)]i, did not block either effect of caffeine. But when the SERCA was inhibited by cyclopiazonic acid the undershoot disappeared. The phosphodiesterase inhibitor IBMX did not influence the undershoot. These results suggest that the undershoot is generated by the Ca(2+)] ATPase of the stores rather than that of the plasma membrane. Since the undershoot increased as [Ca(2+)]i increased, we conclude that at higher levels of [Ca(2+)]i the stores refill more rapidly.     

The effect of fetal calf serum on intracellular calcium in GH3 cells

We have investigated the mechanism of action of fetal calf serum (FCS) on GH3 pituitary tumour cells by measuring intracellular free calcium levels. On the addition of FCS (1%) there was a transient increase in intracellular Ca2+ levels which was attenuated in conditions of reduced extracellular calcium concentrations. The Ca2+ response was abolished by the prior addition of lanthanum chloride (1mM). In contrast, the elevation of cytosolic calcium levels by TRH (100nM), an agonist which causes the mobilisation of calcium from the endoplasmic reticulum, was attenuated but not abolished by lanthanum chloride (1mM). We suggest that FCS (1%) causes the release of calcium from the plasma membrane and the influx of calcium from the extracellular milieu, but does not mobilise calcium from the endoplasmic reticulum.     

The effect of sulfated polysaccharides on the free intracellular calcium ion concentration of lymphocytes

Recent studies have demonstrated that murine lymphocytes express specific cell-surface receptors for a range of sulfated polysaccharides. In order to determine whether polysaccharide binding induces transmembrane signaling, the effects of sulfated polysaccharides on the free intracellular calcium ion concentration [( Ca2+]i) of mouse thymocytes and spleen cells were determined. Cells were loaded with Indo-I, a fluorescent indicator of calcium ion concentration. The validity and limitations in the use of this indicator in the determination of [Ca2+]i are documented. Dextran sulfate (Mn = 500,000), iota-carrageenan, lambda-carrageenan and kappa-carrageenan all cause relatively large changes in the [Ca2+]i of thymocytes (change in [Ca2+]i greater than 50 nM). Of these, dextran sulfate (Mn = 500,000) always had the greatest effect on [Ca2+]i. Smaller responses were obtained with heparin and dextran sulfate (Mn = 5000), while no response was obtained with chondroitin 4-sulfate, chondroitin 6-sulfate, pentosan sulfate or fucoidin. This response pattern (with the exception of fucoidin and pentosan sulfate) corresponds with the expression of thymocyte receptors for these polysaccharides. The increase in [Ca2+]i caused by the sulfated polysaccharides requires extracellular Ca2+ ions however, it is unlikely that voltage-dependent ion channels are involved in these responses. In contrast to thymocytes, although spleen cells express receptors for sulfated polysaccharides, they were unresponsive to all of the sulfated polysaccharides tested, suggesting a basic difference between thymocytes and peripheral T and B lymphocytes in their response to the binding of sulfated polysaccharides.     

Esterification of cholesterol and 25-hydroxycholesterol by rat liver microsomes

The properties of an enzyme in rat liver microsomes was described that catalyzed the formation of 25-hydroxycholesteryl ester in the presence of labeled sterol and oleoyl-CoA. The reaction was similar in several respects to that of cholesteryl ester formation by acyl-CoA: cholesterol acyltransferase. Trypsin pretreatment of microsomes inhibited the esterification of both sterols and a similar dose-dependent inhibition was produced by addition of progesterone and several androgens. Microsomes with an enhanced cholesterol content resulting from in vivo treatment with ethinyl estradiol showed increased esterifying activity towards both cholesterol and 25-hydroxycholesterol. Esterification of endogenous microsomal cholesterol was increased by the addition of 25-hydroxycholesterol, concomitant with 25-hydroxycholesteryl ester formation. To assess the relationship between the association of sterols with membranes and sterol ester formation, microsomes were preincubated with either sterol, reisolated by ultracentrifugation in a density gradient and then analyzed chemically or enzymatically. Cholesterol and 25-hydroxycholesterol both associated with microsomes and the added sterol was subsequently esterified. Maximal esterification was only partially dependent on the amount bound. Progesterone, which inhibited sterol esterification, did not bind to microsomes and no inhibition was observed in reisolated microsomes, indicating that the inhibition produced by progesterone was reversible.     

A comparison of solution properties of cholesterol and 25-hydroxycholesterol

Although 25-hydroxycholesterol is a potent effector of mammalian cellular cholesterol biosynthesis, cholesterol itself is not. In an effort to explain this difference, the critical micellar concentration, liposome-water partition coefficient, and capacity for acyl chain ordering of 25-hydroxycholesterol are measured and compared to those of cholesterol. It is found that 25-hydroxycholesterol differs from cholesterol by existing in monomeric form in aqueous solution at physiologically active concentrations. It is suggested that this property allows 25-hydroxycholesterol but not cholesterol itself to interact in a concentration-dependent fashion with particular proteins.     

The effects of isoproterenol on intracellular calcium concentration

beta-Adrenergic agonist, isoproterenol (ISO), is a potent relaxant of tracheal smooth muscle and inhibits carbachol-induced contraction. The effect of ISO on intracellular free Ca2+ concentration ([Ca2+]i) was examined in bovine tracheal smooth muscle strips, employing aequorin as Ca2+ indicator. Surprisingly, 10 microM ISO induces a 5-fold increase in [Ca2+]i which then gradually declines but still remains higher than basal after 1 h of stimulation. The ISO-induced increase in [Ca2+]i is dose-dependent, and the ED50 is approximately 50 nM. The ISO-induced increase in [Ca2+]i is inhibited by a beta-receptor blocker, propranolol, not by an alpha-blocker, phentolamine. The ISO-induced rise in [Ca2+]i is dependent on extracellular Ca2+. Forskolin, an adenylate cyclase activator, and vasoactive intestinal peptide, which is known to stimulate adenylate cyclase via a specific receptor in this tissue, have similar effects on [Ca2+]i, suggesting that a rise in cyclic AMP concentration mediates this effect of ISO on [Ca2+]i. Pretreatment of muscle with 10 microM ISO inhibits both the initial Ca2+ transient and the contractile response induced by 0.3 microM carbachol. Conversely, in carbachol-pretreated muscle strips, addition of ISO causes a fall rather than a rise in [Ca2+]i, and an inhibition of contraction. These results indicate that ISO has effects on cellular Ca2+ metabolism at more than a single site in bovine tracheal smooth muscle, that these effects are different in control and carbachol-pretreated muscle, and that the relaxing effect of ISO is not due solely to its effect on Ca2+ metabolism.     

Investigation of the effect of intracellular calcium on the cAMP-mediated increase in the calcium current

The experiments were conducted on intracellularly persfused neurons of the molluskHelix pomatia, in which a serotonin (5-HT)-induced increase in the calcium current (I_Ca), mediated by cAMP, is observed. It was established that desensitization of the cell to the action of 5-HT is due to an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). At [Ca²⁺]_i=10^–7 and 10^–6 M, the effect of 5-HT constituted 60 and 32% of this value in the control (in the presence of 10 mM EGTA in the intracellular solution), respectively; at [Ca²⁺]_i=10^–5 M, there was no effect of 5-HT at all. The addition of the calmodulin antagonist trifluoperazine (5·10^–5 mM) or blockers of phosphodiesterase (5 mM theophylline or 10^–4 M IBMX) to the perfusate sharply weakened the ability of calcium to inhibit the effect of 5-HT; at [Ca²⁺]_i=10^–5 M, in the presence of one of these compounds, the effect constituted 60–70% of its control value. It is concluded that the calcium-calmodulin-dependent phosphodiesterase is a key link in the interaction of the two-signal transduction pathway — Ca²⁺ and cAMP in modulation of the calcium-channel activity.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 306–313, May–June, 1991.     

The effect of hyperosmolarity on muscle glycogen accumulation.

Soleus (red) and extensor digitorum longus (white) muscles from Sprague Dawley rats were incubated with 6-14C-labelled glucose in normal and in hyperosmotic media. Hyperosmolarity decreased 6-14C-glucose incorporation into muscle glycogen in a dose dependent manner and increased glycolysis and glucose oxidation. Increased glycogenolysis rather than decreased glycogenesis was responsible for the reduction in labelled glycogen accumulation.     

Mechanism of intracellular calcium transients.

Calcium ions mediate extracellular signals on intracellular processes. The signalling system based on transient rises or oscillations of the cytoplasmic calcium concentration has potential advantages. The relevant mechanisms of intracellular concentration changes include calcium-induced calcium release and calcium dependent inactivation of calcium release. A model has been devised based on these processes to generate repetitive transients of the cytoplasmic calcium concentration.     

On the formation and possible biological role of 25-hydroxycholesterol

The oxysterol 25-hydroxycholesterol is a widely used compound displaying an array of pharmacological actions in in vitro systems and cell based experimental systems. In spite of the frequent use of this compound over the last few decades and a large number of studies in vitro and in vivo, its mechanism of formation in vivo is still not well understood.     

Production of 25-hydroxycholesterol by testicular macrophages and its effects on Leydig cells

Testicular macrophages secrete 25-hydroxycholesterol, which can be converted to testosterone by neighboring Leydig cells. The purposes of the present studies were to determine the mode of production of this oxysterol and its long-term effects on Leydig cells. Because oxysterols are produced both enzymatically and by auto-oxidation, we first determined if testicular macrophages possess cholesterol 25-hydroxylase mRNA and/or if macrophage-secreted products oxidize cholesterol extracellularly. Rat testicular macrophages had 25-hydroxylase mRNA and converted 14C-cholesterol to 14C-25-hydroxycholesterol; however, radiolabeled cholesterol was not converted to 25-hydroxycholesterol when incubated with medium previously exposed to testicular macrophages. Exposure of Leydig cells to 10 microg/ml of 25-hydroxycholesterol, a dose within the range known to result in high basal production of testosterone when tested from 1 to 6 h, completely abolished LH responsiveness after 2 days of treatment. Because 25-hydroxycholesterol is toxic to many cell types at 1-5 microg/ml, we also studied its influence on Leydig cells during 4 days in culture using a wide range of doses. Leydig cells were highly resistant to the cytotoxic effects of 25-hydroxycholesterol, with no cells dying at 10 microg/ml and only 50% of cells affected at 100 microg/ml after 2 days of treatment. Similar conditions resulted in 100% death of a control lymphocyte cell line. These results demonstrate that 1) testicular macrophages have mRNA for cholesterol 25-hydroxylase and can convert cholesterol into 25-hydroxycholesterol, 2) macrophage-conditioned medium is not capable of auto-oxidation of cholesterol, 3) Leydig cells are highly resistant to the cytotoxic influences of 25-hydroxycholesterol, and 4) long-term treatment with high doses of 25-hydroxycholesterol results in loss of LH responsiveness. These results support the concept that testicular macrophages enzymatically produce 25-hydroxycholesterol that not only is metabolized to testosterone by Leydig cells when present at putative physiological levels but also may exert inhibitory influences on Leydig cells when present for extended periods at very high concentrations that may occur under pathological conditions.     

The nonstructural glycoprotein of rotavirus affects intracellular calcium levels.

Rotavirus infection of monkey kidney cells has been reported to result in a significant increase in the concentration of intracellular calcium. This increase in intracellular calcium was associated with viral protein synthesis and cytopathic effects in infected cells. We tested the effect of individual rotavirus proteins on intracellular calcium concentrations in insect Spodoptera frugiperda (Sf9) cells. Insect cells were infected with wild-type baculovirus or baculovirus recombinants that contained an individual rotavirus gene. The cells were harvested at different times postinfection, and the intracellular calcium concentration was measured by using fura-2 as a fluorescent calcium indicator. We found that the concentration of intracellular calcium was increased nearly fivefold in infected Sf9 cells that expressed the nonstructural glycoprotein (NSP4) of group A rotavirus, and this increase in intracellular calcium concentration coincided with NSP4 expression. A similar result was observed in insect cells expressing NSP4 from a group B rotavirus, suggesting the conservation of this function among rotavirus groups. Expression of the other 10 rotavirus proteins or of wild-type baculovirus proteins in Sf9 cells did not significantly increase intracellular calcium levels. These results suggest that the nonstructural glycoprotein NSP4 is responsible for the increase in cytosolic calcium observed in rotavirus-infected cells.     

Polarity in intracellular calcium signaling.

The concentration of free calcium ions (Ca(2+)) in the cytosol is precisely regulated and can be rapidly increased in response to various types of stimuli. Since Ca(2+) can be used to control different processes in the same cell, the spatial organization of cytosolic Ca(2+) signals is of considerable importance. Polarized cells have advantages for Ca(2+) studies since localized signals can be related to particular organelles. The pancreatic acinar cell is well-characterized with a clearly polarized structure and function. Since the discovery of the intracellular Ca(2+)-releasing function of inositol 1,4,5-trisphosphate (IP(3)) in the pancreas in the early 1980s, this cell has become a popular study object and is now one of the best-characterized with regard to Ca(2+) signaling properties. Stimulation of pancreatic acinar cells with the neurotransmitter acetylcholine or the hormone cholecystokinin evokes Ca(2+) signals that are either local or global, depending on the agonist concentration and the length of the stimulation period. The nature of the Ca(2+) transport events across the basal and apical plasma membranes as well as the involvement of the endoplasmic reticulum (ER), the nucleus, the mitochondria, and the secretory granules in Ca(2+) signal generation and termination have become much clearer in recent years.     

The effect of parahydroxylation of diphenylhydantoin on metaphase accumulation.

DPH has a colchicine-like action on metaphase arrest of cultured human lymphocytes. The first step in detoxification of DPH increased its power to accumulate metaphases 3-fold. This hydroxy derivative [5-[4-hydroxyphenyl]-5-phenylhydantoin, HPPH] 3.6 X 10(-4) M was equivalent colchicine 1 x 10(-5) M in its power to inhibit metaphase completion. The effect of HPPH on mitosis was reversible; colchicine effect was not reversed and vincristine effect was partially reversed by washing drug from the medium. Hydroxylation of DPH did not change its inhibition of DNA synthesis and enhanced inhibition of protein synthesis to a minor degree. Detoxification increased the colchicine-like action of DPH.     

Modelling receptor-controlled intracellular calcium oscillators.

This paper presents mathematical models for the hepatocyte calcium oscillator which follow the concepts in a class of informal models developed to account for the striking dependence on the receptor type of several features of the calcium oscillations, in particular the shape and duration of the free calcium transients. The essence of these models is that the transients should be timed by a build-up of activated GTP-binding proteins, which, combined with positive feedback processes and perhaps with cooperative effects, leads to a sudden activation of phospholipase C (PLC), followed by negative feedback processes which switch off the calcium rise and lead to a fall in free calcium back to resting levels. These models predict pulsatile oscillations in inositol (1,4,5)P3 as well as in free calcium. We show that receptor-controlled intracellular calcium oscillators involving an unknown positive feedback pathway onto PLC and negative feedback from protein kinase C (PKC) onto G-proteins and receptors, or negative feedback by stimulation of GTPase activity can simulate many of the features of observed intracellular calcium oscillations. These oscillators exhibit a dependence of frequency on agonist concentration and a dependence of transient duration on receptor and G-protein type. We also show that a PLC-dependent GTPase activating factor (GAF) could provide explanations for some otherwise puzzling features of intracellular calcium oscillations.