Relative specificities of inhibition of acid cholesteryl ester hydrolase and neutral cholesteryl ester hydrolase in cultured rabbit aortic smooth muscle cells by esterastin and cholesteryl oleyl ether.

Rabbit aortic smooth muscle cells in culture were incubated with 0.04-500 M esterastin. Acid cholesteryl ester hydrolase (ACEH) and neutral cholesteryl ester hydrolase (NCEH) activities were inhibited to a comparable degree, with 50% inhibition occurring in the range of 0.4 M esterastin. Cells incubated with cholesteryl oleyl ether showed 50% inhibition of NCEH at 5.0 M, but no inhibition of ACEH over a concentration range of 0.2-20 M. This relative specificity of cholesteryl oleyl ether for NCEH can be employed to study the relative roles of ACEH vs. NCEH in preventing cellular cholesteryl ester accumulation.

Effect of membrane cholesterol enrichment or depletion on the partition behavior of human erythrocytes in dextran-poly(ethylene glycol) aqueous phases.

It has previously been shown that by appropriate manipulation of polymer concentrations and ionic composition and concentration one can select whether charge-associated or lipid-related membrane surface properties are reflected by cell partition in dextran-poly(ethylene glycol) aqueous two-phase systems (Walter, H. (1977) in Methods of Cell Separation ((Catsimpoolas, N., ed.), Vol. 1, pp. 307-354, Plenum Press, New York). In the current experiments we have studied that partition behavior of human erythrocytes and found that not only lipid-related but also charge-associated membrane properties are altered as a consequence of cholesterol-enrichment or -depletion. Results further indicate that, just as cell partition in charged phase systems reflects membrane charge-associated properties not readily measured by means other than partition (Brooks, D.E., Seaman, G.V.F. and Walter, H. (1971) Nat. New Biol. 234, 61--62; Walter, H., Tung, R., Jackson, L.J. and Seaman, G.V.F. (1972) Biochem. Biophys. Res. Commun. 48, 565--571), cell partition in uncharged phases reflects membrane lipid-related properties also not readily measured by other means.

Effects of chronic estrogen replacement therapy on beat-to-beat blood pressure dynamics in healthy postmenopausal women.

Recent data showing gender differences in autonomic control of heart rate and acute estrogen effects on vasodilatation suggest that estrogen may influence autonomic regulation of heart rate and blood pressure. We aimed to determine the effect of postmenopausal estrogen replacement therapy on autonomic control of beat-to-beat heart rate and blood pressure dynamics. Subjects included 20 healthy postmenopausal women aged 60 to 75 years with normal exercise tolerance tests, 10 of whom were taking oral estrogen for 13 +/- 3 (+/- SEM) years. Six healthy premenopausal women were also studied. Continuous electrocardiographic and noninvasive radial artery blood pressure measurements and intermittent forearm blood flow recordings (by venous-occlusion plethysmography) were obtained before and after a 20-minute, 60 degrees head-up tilt and a 420-kcal meal during periods of spontaneous and metronomic breathing (at 0.25 Hz). Low-frequency (0.01- to 0.15-Hz) and high-frequency (0.15- to 0.50-Hz) heart rate and blood pressure spectral powers were compared with a fast Fourier transform. Cardiovascular and heart rate spectral power responses to upright tilt and meal digestion were the same in postmenopausal estrogen users and nonusers. However, during spontaneous breathing the blood pressure spectral power responses to upright tilt and meal ingestion were significantly different between the two groups of women. The low-frequency systolic pressure power response to upright tilt was smaller in estrogen users than nonusers (P = .01). After meal ingestion nonusers had an early postprandial fall (20 to 30 minutes after the meal) and late rise (50 to 60 minutes) in low-frequency systolic and diastolic pressure powers, which were significantly attenuated in estrogen users (P < .02).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of age and gender on autonomic control of blood pressure dynamics.

Both age and gender influence cardiovascular autonomic control, which in turn may influence the ability to withstand adverse cardiac events and respond to orthostatic stress. The purpose of this study was (1) to quantify age- and gender- related alterations in autonomic control of blood pressure (BP) and (2) to examine the impact of these autonomic alterations on BP response to orthostatic stress. We measured continuous BP and R-R intervals and vasoactive peptide levels in the supine and 60 degrees head-up tilt positions during paced respiration (0.25 Hz) in 89 carefully screened healthy subjects (41 men, 48 women, aged 20 to 83 years). Data were analyzed by gender (age adjusted) and by age group (gender adjusted). During tilt, women had greater decreases in systolic BP than men (-10.2+/-2 versus -1.2+/-3 mm Hg; P=0.02) and smaller increases in low-frequency (sympathetically mediated) BP power (P=0.02). Upright plasma norepinephrine was lower in women (P=0.02). Women had greater supine high-frequency R-R interval power than men (P=0.0001). In elderly subjects, the tilt-induced increase in low-frequency BP power was also diminished (P=0.01), despite higher supine (P=0.02) and similar upright norepinephrine levels compared with younger subjects. Thus, healthy women have less sympathetic influence on BP and greater parasympathetic influence on R-R interval than men. Elderly subjects also have reduced sympathetic influence on BP, but this appears to be more consistent with a reduction in vasomotor sympathetic responsiveness.

Intestinal adaptation after jejunoileal bypass in man.

Gastrointestinal anatomy and function has been studied prospectively in 12 patients undergoing jejunoileal bypass surgery in order to investigate the adaptive response of the intestinal mucosa. The total thickness of the jejunal mucosa did not change after surgery, but the crypts became relatively deeper, suggesting a more rapid turnover of gastrointestinal cells. The absorption of oxalate was depressed in the immediate postoperative period but had improved toward preoperative levels by 6 months. Vitamin B12 absorption also declined postoperatively, and increased thereafter in the patients with an end-to-end jejunoileostomy, but showed a much smaller recovery in the group with an end-to-side anastomosis. The cholesterol concentration (lithogenicity) of the duodenal bile rose by 30% in the first 3 weeks after surgery, but had returned to preoperative levels by 6 months. The segmental absorption of glucose across the jejunum declined after surgery. Caloric intake also declined, whether measured as the quantity of food that patients elected to eat over a 24-hr period, or as the quantity of a liquid lunch which they consumed over a 20-min period. The level of basal gastric acid was increased postoperatively but the maximal output after histamine stimulation was not. The gastrin response to a standard liquid meal was also significantly increased after surgery. Enteroglucagon secretion showed an increase in 3 weeks and a further increase by 6 months after intestinal bypass surgery. The significance of these changes to intestinal adaptations is discussed.

Comparison of acetate-1-14C metabolism in uremic and nonuremic dogs.

Acetate-1-14C was infused into six anephric uremic and six anephric nonuremic dogs during a 4-hr hemodialysis against a standard acetate containing (39.5 mM) dialysis solution. Arterial acetate (nonradioactive) levels achieved a steady state by the end of dialysis indicating that the maximum rate of acetate metabolism had not been exceeded. The mean arterial acetate level at the end of dialysis was 2.6 mM in both groups of dogs. Acetate disappearance after the cessation of dialysis followed first order kinetics with a mean half-life of 3.8 +/- 0.5 min in the uremic and 3.7 +/- 0.5 min in the nonuremic dogs. Most of the infused acetate-1-14C was metabolized to 14CO2 within 8 hr after dialysis. An average of 84 and 71% of the infused acetate-1-14C was metabolized to 14CO2 in the uremic and nonuremic dogs, respectively. Small but significant amounts of radioactivity were incorporated into lipids of plasma and other tissues. Incorporation of radioactivity into total lipids of liver, omental fat, and sciatic nerve was significantly greater in the uremic as compared to the nonuremic dogs. Incorporation of radioactivity into total lipids of heart, aorta, and plasma was the same in both groups of dogs.

Cholesterol oxidation derivatives and arterial endothelial damage.

Three groups of New Zealand male while rabbits were given either 2.5 mg/kg of 25-hydroxycholesterol, cholestane-3 beta, 5 alpha, 6 beta-triol or vehicle only, intravenously. 24 h after treatment, the luminal surfaces of aortae of rabbits receiving 25-hydroxycholesterol were examined by scanning electron microscopy (SEM) and showed numerous balloon-like protrusions and crater-like defects as well as circulating, formed elements adhering on the luminal surface. The luminal surface of aortae of rabbits given cholestane-3 beta, 5 alpha, 6 beta-triol had similar but more frequent lesions when compared with those of the 25-hydroxycholesterol group. Microthrombi were occasionally found. The aortae of the control group had significantly fewer lesions. Transmission electron-microscopic studies showed intracytoplasmic vacuoles and diffuse subendothelial edema in the aortae of the two groups receiving the oxidation derivatives of cholesterol. The balloon-like protrusions and crater-like defects observed by SEM appeared to represent the initial sterol-induced endothelial cell injury. Repeated episodes of arterial injury followed by thrombus formation could eventually lead to atherosclerosis.

Transport of cholesterol autoxidation products in rabbit lipoproteins.

Radiolabeled pure [4-14C]cholesterol was kept at 60 degrees C under air to autoxidize for 5 weeks, after which approximately 12% cholesterol oxidation products were formed. The mixture, suspended in gelatin, was given to rabbits by gastric gavage. Rabbits were killed 4, 24, and 48 h after treatment. Cholesterol and its autoxidation products were separated by thin-layer chromatography into 5 fractions and radioactivities of each fraction were measured. Percentages of each fraction of cholesterol oxidation products and cholesterol in the original mixture before administration and in the rabbit sera after administration were similar, suggesting that the rates of absorption of cholesterol oxidation products are not significantly different from that of cholesterol. Lipoproteins were fractionated by ultracentrifugation into VLDL, LDL and HDL. Radioactivities of each fraction in lipoproteins separated by thin layer chromatography showed that fractions containing cholestane-3 beta,5 alpha,6 beta-triol, 7 alpha- and 7 beta-hydroxycholesterol and 7-ketocholesterol were more selectively transported in VLDL, whereas most of the 25-hydroxycholesterol was present in LDL. HDL contained only minute amounts of cholesterol oxidation products.

Relationships of adenine nucleotide metabolism to platelet-collagen adhesion.

Adhesion of platelets to collagen fibrils in a stirred system was inhibited by preincubation of platelets with combinations of 2-deoxy-D-glucose and oligomycin or antimycin. The inhibition of adhesion was associated with a decrease in metabolic ATP to 6% of control levels. Without metabolic inhibitors, platelets adherent to collagen fibrils were found to have catabolized approximately 57% of their metabolic ATP, and converted a major part of this to IMP. Storage pool ATP and ADP contents were also diminished in the adherent platelets. Pretreatment with imipramine resulted in 76% inhibition of the release reaction, but only 5% inhibition of adhesion. Imipramine-treated platelets that were adherent to collagen showed significant depletion of metabolic ATP, but markedly diminished conversion of ATP to IMP as compared to control adherent platelets. Inhibition of deamination of platelet AMP by coformycin or erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) did not inhibit adhesion, although platelets adherent to collagen after treatment with these agents showed depletion of metabolic ATP. These studies suggest that adhesion is an energy dependent process, occurring independently of release, and not associated with conversion of ATP to IMP. The energy dependent portions of the adhesion process are probably disc to sphere transformation and pseudopod formation, the ATP threhold requirement is relatively low, and the ATP utilized can probably be regenerated during the adhesion process via glycolysis and oxidative phosphorylation.

Cholesterol ester hydrolysis by homogenates of whole testis, seminiferous tubules and interstitial cells of mature rats.

The characteristics and localization of a cholesterol ester hydrolase enzyme in homogenates of whole testis and in isolated seminiferous tubules and interstitial cells of mature rats have been investigated. Hydrolysis of cholesteryl [1(-14)C]oleate occurred at an optimum pH of 7.0, was linearly related to time up to 5--6 h of incubation and increased linearly up to 0.25 mg protein/incubation. Hydrolytic activity was inhibited by increasing the incubation temperature from 29 to 41 degrees C and by sonication. Cholesterol ester hydrolase activity/mg protein was three times greater in homogenates of seminiferous tubules than in interstitial cells. Cholesterol ester hydrolase may function to provide precursors for use in seminiferous tubular steroid hormone biosynthesis or germ cell maturation.

Comparison of several activated partial thromboplastin time methods.

Activated partial thromboplastin times (APTT's) performed with a semi-automated electrical-conductivity type of clot timer on plasmas from patients with hepatic disease and intravascular coagulation, and on warfarin or heparin therapy, were significantly lower than when done on the same plasmas with either a manual optical method or an automated optical-endpoint instrument. Results of APTT's done on normal plasmas by the three methods were not significantly different. Substitution of different activator-phospholipid reagents resulted in some variability in results, but these differences were less than those between the different done with both the electrical clot timer and the automated optical instrument on prepared plasmas containing 5.0 or 1.0% of factor II, V, VIII, IX, OR X revealed shorter times with the electrical clot timer only in the case of factor II- and factor V-deficient plasmas. APTT's done on normal plasmas to which 0.1 or 0.3 units per ml. of heparin had been added vitro also were shorter with the electrical clot itmer than the automatic optical instrument. Prothrombin times done on normal and abnormal control plasmas and on a series of plasmas from patients on warfarin therapy showed no significant difference between the two methods.

Effects of cholesterol oxides on LDL receptors in cultured rabbit aortic smooth muscle cells.

The efficiency of receptor-mediated low-density lipoprotein (LDL) uptake and degradation has been implicated in the development of atherosclerosis. It has previously been shown that cholesterol oxides have more effect on the feedback control of cholesterol biosynthesis than does cholesterol per se on a molecule-for-molecule basis. Cholesterol oxides may also modify the expression or function of the LDL receptors, resulting in alteration in cholesterol homeostasis. Rabbit aortic smooth muscle cells were preincubated in 5% lipoprotein-deficient medium for 24 hours and then incubated with 1 or 5 µg/ml purified cholesterol or cholesterol oxides, including 25-hydroxycholesterol, 7-ketocholesterol, cholestane-3ß,5α,6ß-triol, and cholesterol 5α, 6α-epoxide for 12 to 24 hours. The uptake of (125)I-LDL was significantly suppressed in a dose-dependent fashion to 67% of the control value by 25-hydroxycholesterol at 1 µg/ml and to 53% at 5 µg/ml after the 24-hour incubation period. Other cholesterol oxides also had inhibitory effects on the LDL uptake at 5 µg/ml. At these concentrations cholesterol oxides additionally inhibited cellular degradation of LDL and the fractional turnover rate of LDL was prolonged.

Effect of uremia on incorporation of acetate into rat plasma and tissue lipids.

Experimental uremia was induced in rats by means of bilateral nephrectomy or bilateral ureteral ligation. Incorporation of acetate-1-14C into expired 14CO2 and into plasma and tissue lipids was studied immediately after surgery and at 48 hr, when the rats were uremic. In rats studied immediately after surgery, bilateral nephrectomy, but not bilateral ureteral ligation, significantly decreased the conversion of acetate-1-14C into expired 14CO2. In uremic rats at 48 hr, acetate-1-14C metabolism to 14CO2 was not significantly altered in either group. Plasma triglyceride concentrations and 14C-acetate incorporation into triglycerides were increased in the 48-hr uremic groups, but plasma and liver triglyceride specific radioactivities were not significantly altered. Plasma free fatty acid concentrations and incorporation of acetate into free fatty acids were lower in the 48-hr uremic groups than in controls. Plasma cholesterol concentrations and specific radioactivities were increased in these uremic groups, as were liver free cholesterol specific activities. These results suggest that increased triglyceride and cholesterol synthesis from acetate may contribute to the hypertriglyceridemia and hypercholesterolemia observed in uremic rats.

Inconsistent link between low-frequency oscillations: R-R interval responses to augmented Mayer waves.

Low-frequency oscillations in arterial blood pressure (Mayer waves) and R-R interval are thought to be linked through the arterial baroreflex. To delve into this relationship, we applied low (10 mmHg) and moderate (30 mmHg) lower body negative pressure (LBNP) in 10-s cycles to 18 healthy young male subjects. They showed no change in average blood pressure with this oscillatory stimulus but did show a significant decrease in R-R interval (P < 0.05) during both levels of LBNP. In addition, we succeeded in augmenting low-frequency blood pressure oscillations in a graded response to oscillatory LBNP level (P < 0.05) while significantly increasing low-frequency R-R interval oscillations (P < 0.05). However, cross-spectral coherence between these increased oscillations was highly variable across individuals and stimulus level. Although nearly all subjects showed significant coherence during basal conditions (n = 17), only seven subjects maintained significant coherence during both levels of LBNP. These results suggest that a complex interaction of regulatory mechanisms determines the link between low-frequency oscillations and the responses to even low levels of LBNP.

Lipid composition of bile in morbid obesity before and after jejunoileal bypass surgery.

Bile cholesterol, phospholipids, total bile acids, individual bile acids, and fatty acid compositions of bile neutral lipids and phospholipids were analyzed before, at one month and at six months following jejunoileal bypass surgery in a series of morbidly obese patients. Preoperative mole percentages of cholesterol and lithogenic indices were high, indicating that biles were supersaturated with cholesterol and outside the micellar solubility zone when plotted on triangular coordinates. At the one month post-operative period percentages of cholesterol and lithogenic indices were significantly increased as compared to the pre-operative state. At six months post-operatively these values had decreased to approximately the pre-operative levels. No changes were observed in percentages of lithocholic acid, but deoxycholic acid decreased to markedly low levels at one month and remained low at the six month post-operative interval. Relative proportions of cholic acid increased, and the ratio of cholic to chenodeoxycholic acid was significantly increased at both post-operative intervals. No significant changes were noted in bile neutral lipid or phospholipid fatty acid composition, indicating that no depletion of essential fatty acids had occurred.

A lipoprotein independent assay for human serum lecithin-cholesterol acyltransferase.

A method has been developed for estimation of human serum lecithin-cholesterol acyltransferase free of interference by endogenous lipoproteins. Precipitation of serum low and very low density lipoproteins by sodium phosphotungstate and magnesium chloride results in complete recovery of lecithin-cholesterol acyltransferase activity in the supernatant. One microliter of the supernatant can be accurately assayed with a highly efficient substrate containing phosphatidylcholine-cholesterol vesicles and apo-high density lipoproteins (HDL), with no interference from endogenous HDL or residual precipitation reagents. Serum levels of the enzyme were found to be reduced in patients with parenchymal liver disease, renal disease, gastrointestinal tumors and anemias.

Effects of administration of hypolipidemic agent, 2,2'''- [ (1-methyl-4, 4-diphenylbutylidene) bis (p-phenyleneoxy)] bistriethylamine oxalate (SQ 10,591) upon cholesterol esterification by aorta, adrenal, and testes of cholesterol-fed rabbits].

Administration of 2,2'''-([1-methyl-4, 4-diphenylbutylidene] bis(p-phenyl-eneoxy]) bistriethylamine oxalate (SQ 10,591) at 20 mg/Kg daily for 5 days to cholesterol-fed rabbits resulted in no change in aortic microsomal cholesterol esterification with a palmitoyl coenzyme A substrate or of aortic mitochondrial cholesterol esterification with a palmitate substrate. Esterification by both reactions in the adrenal was much higher than in either aorta or testes. Adrenal and testicular mitochondrial esterification and testicular microsomal esterification were inhibited significantly after SQ 10,591 administration. In vitro addition of 0.0001 M SQ 10,591 significantly inhibited both microsomal and mitochondrial cholesterol esterification in aorta, adrenal, and testes.

Effects of cholesterol oxidation derivatives on cholesterol esterifying and cholesteryl ester hydrolytic enzyme activity of cultured rabbit aortic smooth muscle cells.

The effects of 5 micrograms/ml of 25-hydroxycholesterol; cholestane-3 beta,5 alpha,6 beta-triol; and cholesterol on acyl CoA cholesterol acyltransferase, acid cholesteryl ester hydrolase and neutral cholesteryl ester hydrolase was studied in cultured rabbit aortic smooth muscle cells. After 1 hour incubation, 25-hydroxycholesterol resulted in a fourfold stimulation of acyl CoA cholesterol acyltransferase activity. No stimulation by 25-hydroxycholesterol was noted before 15 minutes or after 5 hours of incubation. Neither cholestane-3 beta,5 alpha,6 beta-triol nor cholesterol influenced acyl CoA cholesterol acyltransferase activity at any time interval. No significant effects of any of the sterols were noted on acid cholesteryl ester hydrolase or neutral cholesteryl ester hydrolase activity. The imbalance between acyl CoA cholesterol acyl transferase and hydrolase activities induced by 25-hydroxycholesterol could result in cholesteryl ester accumulation by arterial smooth muscle cells, which may be associated with atherosclerosis.

In vitro incorporation of acetate-1-(14)C into the phospholipids of rabbit and human endometria.

Endometria from nonpregnant and 6-day pregnant rabbits and from humans in the proliferative and secretory phases were incubated with 1-(14)C-acetate.(14)CO(2) was collected, and subsequently the amounts, specific radioactivities, and in some cases the fatty acid compositions of the isolated phospholipids were determined. Phosphatidyl choline was the phospholipid present in highest amount in endometria from both nonpregnant and pregnant rabbits, and in human endometria; this phospholipid also showed the highest degree of incorporation of(14)C-acetate. Pregnancy in the rabbit seemed to decrease the incorporation of(14)C-acetate into most of the endometrial phospholipid classes. In humans, the incorporation of acetate into phosphatidyl choline and phosphatidyl ethanolamine was lower in the secretory than the proliferative endometria.Of the fatty acids, linoleic acid in phosphatidyl choline and phosphatidyl ethanolamine of the rabbit endometria showed a significant relative increase during pregnancy and palmitoleic acid showed a decrease.

Inhibition of human lecithin cholesterol acyltransferase by monoterpenes.

The lecithin-cholesterol acyltransferase activity of human plasma was found to be inhibited by Rowachol, a proprietary mixture of pure monoterpenes. Menthol, the major ingredient in Rowachol (32%), and a number of other monoterpenes were found to inhibit the enzyme independently. Concentrations of monoterpenes required to achieve 50% inhibition were of the same order of magnitude as the cholesterol concentration present in the reaction mixture.