Instrumental learning of vasomotor responses by rats: learning to respond differentially in the two ears.

Curarized and artificially respirated rats were rewarded by electrical stimulation of the brain for changes in the balance of vasomotor activity between the two ears. They learned vasomotor responses in one ear that were independent of those in the other ear, in either forepaw, or in the tail, or of changes in heart rate or temperature. In addition to implications for learning theory and psychosomatic medicine, these results indicate a greater specificity of action in the sympathetic nervous system than is usually attributed to it.

Unexpected adrenergic effects of chlorpromazine: eating elicited by injection into rat hypothalamus.

Although chlorpromazine is believed to block adrenergic transmission, injection of this drug into the hypothalamus of satiated rats does not block norepinephrine-elicited eating, but instead mimics norepinephrine by eliciting eating. The amount of eating elicited by norepinephrine and by chlorpromazine is reliably correlated. These results suggest that endogenous norepinephrine mediates eating elicited by centrally injected chlorpromazine.

Suppression of food intake with intragastric loading: relation to natural feeding cycle.

Rats were infused through chronically implanted intragastric tubes with 100 percent of their normal total daily food intake. The infusion was given either continuously over 24 hours or divided into discrete meals programed to simulate the rats' natural eating pattern. The same diet was also available for consumption by mouth. In neither case did the animals completely stop eating. During slow infusions excessive consumption ranged from 30 to 50 percent. During simulated meal infusion of the same total quantity of diet, they compensated far better, overeating by only 2 to 18 percent. Periodic filling of the stomach between scheduled meals was no more effective than a continuous slow infusion. Therefore, factors related to the natural feeding cycle make a significant contribution to the effectiveness of food in maintaining satiety and controlling food intake.

Suppression of immunity by stress: effect of a graded series of stressors on lymphocyte stimulation in the rat.

In rats a graded series of stressors produced progressively greater suppression of lymphocyte function, as measured by the number of circulating lymphocytes and by phytohemagglutinin stimulation of lymphocytes in whole blood and isolated cultures. This evidence suggests that stress suppresses immunity in proportion to the intensity of the stressor.

Stress-induced suppression of immunity in adrenalectomized rats.

Stress-induced suppression of lymphocyte stimulation by phytohemagglutinin was demonstrated in Isolated lymphocytes and in cultures of whole blood from adrenalectomized rats. The results demonstrate that corticosteroid independent mechanisms participate in the suppression of lymphocyte function by stressors. Stress-induced lymphopenia, however, was found to be adrenal dependent, indicating that the modulation of immunity by stress is complex and multidetermined.

Baroreceptor activation reduces reactivity to noxious stimulation: implications for hypertension.

The hypothesis was tested that an acute rise of blood pressure may reduce reactivity to noxious stimuli through a baroreceptor-mediated reduction of cerebral arousal. When blood pressure was raised by an infusion of phenylephrine, rats showed less running to terminate or avoid noxious stimuli than during saline infusions. This effect was not seen in rats with denervated baroreceptors. The results suggest that a rise of blood pressure could have motivational consequences significant for human hypertension.

Individual variation in the effects of dietary cholesterol on plasma lipoproteins and cellular cholesterol homeostasis in man. Studies of low density lipoprotein receptor activity and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in blood mononuclear cells.

The effects of dietary cholesterol on plasma lipoproteins and cholesterol homeostasis in blood mononuclear cells have been examined in healthy adults. Addition of 1,500 mg of cholesterol to the daily diet of 37 subjects for 14 d was associated with a wide range of response of plasma total cholesterol concentration (from -6 to +75 mg/dl; mean change, +29 mg/dl; P < 0.001). Increases in plasma cholesterol reflected increased cholesterol concentrations in intermediate density lipoprotein (IDL; 1.006-1.019 g/ml), low density lipoprotein (LDL; 1.019-1.063 g/ml), and the HDL(2) subclass (1.063-1.125 g/ml) of high density lipoprotein, which on average accounted for 20, 58, and 22%, respectively, of the total increment. Similar responses occurred in 14 other subjects given 750 mg cholesterol per day for 28 d. Plasma apolipoprotein B concentrations in IDL and LDL also increased. THESE EFFECTS ON PLASMA LIPOPROTEINS WERE ACCOMPANIED BY THREE CHANGES IN FRESHLY ISOLATED BLOOD MONONUCLEAR CELLS: (a) an increase in cell cholesterol content (mean change, +17%; P < 0.01); (b) suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity (-32%; P < 0.001); and (c) reduction of LDL receptor activity (-74%; P < 0.01), quantified as the rate of degradation of (125)I-LDL to noniodide trichloroacetic acid-soluble material. These results provide the first direct evidence for the modulation of LDL receptor activity and HMG CoA reductase activity in a peripheral cell type in response to a dietary perturbation of human lipoprotein metabolism.The percentage increase in LDL cholesterol was negatively correlated with the percentage decrease in HMG CoA reductase activity (r = -0.49, P < 0.01). An additional negative correlation existed between the increment in plasma cholesterol concentration and the capacity of cells to degrade (125)I-LDL after derepression by preincubation for 72 h in lipoprotein-deficient medium (r = -0.74, P < 0.001). Thus, differences between individuals in the responses of the plasma lipoproteins to dietary cholesterol appear to be related in part to differences in the capacity of peripheral cells to catabolize LDL and to down-regulate cholesterol synthesis.

Interaction between high density and low density lipoproteins uptake and degradation by cultured human fibroblasts.

High density lipoprotein (HDL) inhibited the binding (trypsin-releasable radioactivity), internalization (cell-associated radioactivity after trypsinization), and degradation (TCA-soluble non-iodide radioactivity) of (125)I-low density lipoprotein ((125)I-LDL) by cultured normal human fibroblasts. At HDL:LDL molar ratios of 25:1 (protein ratios about 5:1), these parameters were reduced by about 25%. Unlabeled LDL was about 25 times more effective in reducing (125)I-LDL binding, implying that if HDL and LDL bind at common sites the affinity of HDL for these sites is very low or that the interaction is on some other basis. The fractional reduction in (125)I-LDL binding at a given HDL: (125)I-LDL ratio was independent of (125)I-LDL concentration and occurred equally with fibroblasts from a subject with homozygous familial hypercholesterolemia. Reciprocally, the binding, internalization, and degradation of (125)I-HDL were reduced by LDL. Preincubation of fibroblasts with HDL (or LDL) reduced the subsequent binding of (125)I-LDL (or (125)I-HDL) during a second incubation. In other studies HDL reduced the net increase in cell cholesterol content induced by incubation with LDL. HDL alone had no net effect on cell cholesterol content. These findings suggest that HDL reduces both the high affinity and the low affinity binding of LDL to human fibroblasts and that this in turn reduces the internalization and degradation of LDL. The effect of HDL on the LDL-induced changes in cell cholesterol content could be in part on this basis and in part on the basis of an HDL-stimulated release of cholesterol from the cells. These effects of HDL in vitro may be relevant to the negative correlations reported from in vivo studies between plasma HDL concentration and both body cholesterol pool size and the prevalence of clinically manifest atherosclerosis but further studies will be needed to establish this.

Splanchnic metabolism of plasma apolipoprotein B: studies of artery-hepatic vein differences of mass and radiolabel in fasted human subjects.

The metabolism of apoprotein B-containing plasma lipoproteins by human splanchnic tissues has been studied in 29 men undergoing coronary angiography. Before catheterization autologous radio-iodinated lipoproteins were infused into a peripheral vein: 10 subjects received (125)I-labeled Sf 12-60 lipoproteins; 12 received (125)I-labeled Sf 12-60 plus (131)I-labeled Sf 100-400 lipoproteins; and 7 received (125)I-labeled Sf 12-60 plus (131)I-labeled Sf 0-12 lipoproteins. Paired arterial and hepatic vein blood samples were subsequently collected for replicate measurements of apoprotein B (apo B) mass, radioactivity and specific activity in each lipoprotein class. Splanchnic plasma flow was measured with indocyanine green. All studies were conducted after a 14-h overnight fast. Newly synthesized apo B was shown to be secreted by splanchnic tissues as a component of Sf 100-400 lipoproteins, with no detectable uptake of apo B from this class. Sf 12-60 apo B was extracted by the splanchnic bed, with no detectable secretion. After continuous intravenous infusion of (125)I-labeled Sf 12-60 for five or more hours, 41-67% (mean 55%) of extracted Sf 12-60 apo B radioactivity reappeared in hepatic vein Sf 0-12 apo B. There was no detectable splanchnic catabolism of Sf 0-12 apo B. The rates of Sf 100-400 apo B secretion, calculated as the product of artery-hepatic vein concentration difference and splanchnic plasma flow, were greater than the previously reported rates of very low density lipoprotein apo B turnover in fed subjects obtained by kinetic analysis of plasma specific radioactivity decay curves, suggesting that there may be a diurnal variation in hepatic apo B synthesis. They also exceeded the splanchnic extraction rates of Sf 12-60 apo B, suggesting there was some extrasplanchnic catabolism of the apo B of Sf > 60 lipoproteins.

Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods.

A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S:-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously well- studied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships.

Induction of low density lipoprotein receptor synthesis by high density lipoprotein in cultures of human skin fibroblasts.

Further studies have been made of the effects of high density lipoprotein (HDL) on the surface binding, internalization and degradation of 125I-labeled low density lipoprotein (125I-labeled LDL) by cultured normal human fibroblasts. In agreement with earlier studies, during short incubations HDL inhibited the surface binding of 125I-labeled LDL. In contrast, following prolonged incubations 125I-labeled LDL binding was consistently greater in the presence of HDL. The increment in 125I-labeled LDL binding induced by HDL was: (a) associated with a decrease in cell cholesterol content; (b) inhibited by the addition of cholesterol or cycloheximide to the incubation medium; and (c) accompanied by similar increments in 125I-labeled LDL internalization and degradation. It is concluded that HDL induces the synthesis of high affinity LDL receptors in human fibroblasts by promoting the efflux of cholesterol from the cells.

Human hepatic low-density lipoprotein receptors: associations of receptor activities in vitro with plasma lipid and apolipoprotein concentrations in vivo.

The relationships of plasma lipid and apolipoprotein (apo) concentrations to hepatic low-density lipoprotein (LDL) receptor activity were examined in 21 subjects (16 females, 5 males), who were undergoing laparotomy for non-neoplastic disease (cholecystectomy in 16). None had familial hypercholesterolemia, or renal, endocrine or hepatic disease. Ages were 37-77 years (mean, 58 years), plasma cholesterol concentrations 4.09-6.72 mmol/l (5.38) and plasma triacylglycerol concentrations 0.75-2.35 mmol/l (1.36). Receptor activity was quantified in vitro as the total saturable binding and EDTA-suppressible binding (representing apoB,E receptors) of 125I-labelled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C. There were no significant differences between men and women in 125I-labeled LDL binding. In the pooled data, EDTA-suppressible binding averaged 50 ng 125I-LDL protein/mg cell protein (S.D., 15). Total saturable binding averaged 2-fold greater (mean, 101 ng/mg; S.D., 32). Plasma cholesterol, LDL cholesterol and apoB concentrations were negative functions of both EDTA-suppressible binding and total saturable binding, but the correlations with EDTA-suppressible binding were stronger (cholesterol: r = -0.59, P less than 0.01; LDL cholesterol: r = -0.48, P less than 0.05; apoB: r = -0.61, P less than 0.01). Plasma triacylglycerol, high-density lipoprotein cholesterol and apoA-I concentrations were not related to either measure of receptor activity. These results provide evidence that the activity of apoB,E receptors in the liver is a major determinant of the plasma LDL concentration in middle-aged and elderly humans.

Sex difference in the saturable binding of low-density lipoprotein by liver membranes in ageing rats.

It is well documented that women of child-bearing age tend to have lower serum low-density lipoprotein (LDL) concentrations than men. In order to explore the metabolic basis of this sex difference, we have compared the saturable binding of 125I-labeled LDL (d 1.02-1.05 g/ml) at 37 degrees C by liver membranes from healthy male and female Wistar rats of different ages (15-213 days). Woolf plots of saturable binding curves over the concentration range 15-65 micrograms LDL protein/ml were linear and compatible with a single class of binding sites. Maximum binding capacity (Bmax) was not significantly different in male and female animals of 15-19 days of age (respectively, 0.331 +/- 0.018 vs. 0.427 +/- 0.044 micrograms LDL protein/mg membrane protein, mean +/- S.E.). Thereafter, Bmax increased in females, reaching a peak of 0.635 +/- 0.042 micrograms LDL protein/mg membrane protein at 60 days. As no increase in Bmax occurred in males, values were significantly higher (P less than 0.02) in females than in males (by a mean of 61-117%) at all ages after 30 days. During ageing, serum cholesterol concentration changed reciprocally with Bmax in females (Pearson's correlation coefficient, r = -0.761, P less than 0.01) and remained essentially constant in males. The equilibrium dissociation constant for 125I-labelled LDL binding to the hepatic membranes was unaffected by both age and sex. These results provide evidence that the sex difference in the plasma total and LDL cholesterol concentrations is related, at least in part, to a greater mean LDL receptor density in the livers of females.

The very-high-density lipoprotein fraction of rabbit plasma is rich in tissue-derived cholesterol.

When plasma from rabbits, which several weeks earlier had been infused with [3H]cholesterol, was subjected to equilibrium density gradient ultracentrifugation, the specific radioactivity of cholesterol in the very-high-density lipoprotein (VHDL) fraction (d 1.22-1.32 g/ml) was three to 8-fold greater (mean, 5.5-fold; P less than 0.001) than that in high-density lipoproteins (HDL; d 1.06-1.21 g/ml). On size exclusion chromatography of plasma, no increase in specific radioactivity was seen in particles smaller than HDL. These findings suggest that those apolipoprotein-lipid complexes that dissociate from HDL during ultracentrifugation to form the VHDL fraction contain proportionately more tissue-derived cholesterol than do those that are more tightly bound to HDL.

Effects of microtubule-disruptive and membrane-stabilizing agents on low density lipoprotein metabolism by cultured human fibroblasts.

The cellular mechanisms involved in the uptake and metabolism of low density lipoprotein (LDL) by cultured normal human fibroblasts have been investigated with the aid of drugs known to disrupt cytoplasmic microtubules or to inhibit membrane fusion. Two drugs which disrupt microtubules by differing mechanisms, colchicine and vinblastine, each reduced the high affinity surface binding of 125I-labelled LDL by fibroblasts. Associated reductions of the endocytosis and degradation of the lipoprotein could be attributed almost entirely to this effect. In contrast, lumicolchicine, an analogue of colchicine without microtubule-disruptive activity, had little or no effect on 125I-labelled LDL metabolism. Each of two groups of membrane-stabilizing agents, the phenothiazines and the tertiary amine local anaesthetics, directly inhibited both the internalization of 125I-labelled LDL following high affinity binding to cell surface receptors and the catabolism of the lipoprotein subsequent to endocytosis, supporting previous morphological evidence for the importance of membrane fusion in these processes.

Effects of cytochalasin B on low-density lipoproteins metabolism by cultured human fibroblasts.

The role of cytoplasmic microfilaments in the metabolism of low-density lipoprotein by human fibroblasts was studied with the aid of cytochalasin B. At concentrations of 5--40 nmol/ml cytochalasin increased the surface binding but decreased the endocytosis of 125I-labelled low-density lipoprotein. Subsequent studies indicated that these changes reflected a reduction of the rate of internalisation of low-density lipoprotein receptors. Independent inhibitory effects were also observed on low-density lipoprotein degradation and on the cellular release of the trichloroacetic acid-soluble degradation products.

Hormonal determinants of apolipoprotein B,E receptor expression in human liver. Positive association of receptor expression with plasma estrone concentration in middle-aged/elderly women.

The relationships of the expression of hepatic low-density lipoprotein (LDL) receptors (apo B,E receptors) to several plasma hormone concentrations were examined in 15 fasted women aged 37-75 years (mean, 57 years), who were undergoing laparotomy for non-neoplastic disease. No subject had clinical or biochemical evidence of familial hypercholesterolemia, renal disease, hepatic disease, or endocrine disease. Hepatic apo B,E receptor expression was quantified in vitro as the EDTA-suppressible binding of 125I-labeled human LDL (15 micrograms protein/ml) by liver homogenate at 37 degrees C; values were 23-75 ng LDL protein/mg cell protein (mean, 47 ng/mg). Receptor expression was strongly correlated with plasma estrone concentration (rs = +0.70, P = 0.035), but was unrelated to the concentrations of testosterone, thyroxine, free triiodothyronine, cortisol, sex hormone-binding globulin (SHBG) or cortisol-binding globulin. Insulin and estradiol concentrations were mostly very low. The correlation of receptor expression with plasma total estrone concentration reflected associations with both the albumin-bound (rs = +0.78, P = 0.014) and unbound (rs = +0.80, P = 0.009) fractions, but not with the SHBG-bound fraction (rs = -0.22, P = 0.574), of this hormone. As the non-SHBG-bound fractions of gonadal steroids are considered to be the biologically active components, these results are consistent with experimental evidence that the synthesis of apo B,E receptors in hepatocytes is stimulated by estrogens, and suggest that circulating estrone may be the major hormonal determinant of receptor expression in fasted middle-aged/elderly women.

Elevation of plasma high-density lipoprotein concentration reduces interleukin-1-induced expression of E-selectin in an in vivo model of acute inflammation.

BACKGROUND: Although there is strong evidence that plasma HDL levels correlate inversely with the incidence of coronary artery disease, the precise mechanism(s) for the protective effect of HDLs remains unclear. We recently showed that HDLs inhibit endothelial cell expression of cytokine-induced leukocyte adhesion molecules in vitro. Our study therefore sought to test the hypothesis that elevating the level of circulating HDLs would inhibit endothelial cell activation in vivo. METHODS AND RESULTS: We used a porcine model of inflammation previously established in our laboratory, in which the level of vascular endothelial cell expression of E-selectin in interleukin (IL)-1alpha-induced skin lesions was measured by the uptake of a radiolabeled anti-E-selectin antibody (1.2B6). Porcine plasma HDL levels were elevated by use of a bolus injection of reconstituted discoidal HDL (recHDL). These particles resemble nascent HDL particles in shape and contain apolipoprotein A-I as the sole protein and soybean phosphatidylcholine as the sole phospholipid. We found that recHDLs inhibited the expression of IL-1alpha-induced E-selectin by porcine aortic endothelial cells in vitro, confirming that the inhibitory effect is conserved with synthetic HDLs and demonstrating that the phenomenon is not restricted to human endothelial cells. In vivo, elevating the circulating level of HDLs approximately 2-fold led to significant inhibition of basal and IL-1alpha-induced E-selectin expression by porcine microvascular endothelial cells. CONCLUSIONS: These observations demonstrate the potential anti-inflammatory action of HDLs and provide support for the further investigation of the mechanisms underlying the inhibitory effects of HDLs on endothelial cell activation.

Concentrations of electrophoretic and size subclasses of apolipoprotein A-I-containing particles in human peripheral lymph.

When cultured cells are exposed to plasma, the initial acceptors of unesterified cholesterol are small lipid-poor apolipoprotein A-I (apoA-I)-containing high density lipoproteins (HDLs) with pre-beta electrophoretic mobility. These are converted by lecithin:cholesterol acyltransferase into larger spheroidal cholesteryl ester-rich HDLs with alpha mobility. To study the determinants of the concentration of small pre-beta HDLs in tissue fluids, we collected prenodal peripheral lymph from 34 fasted normal men. By crossed immunoelectrophoresis, the concentration of pre-beta HDLs in lymph averaged 20% of that in plasma. On multiple regression analysis, pre-beta apoA-I concentration in lymph was directly related to pre-beta apoA-I concentration in plasma and independently to alpha apoA-I concentration in lymph. Similar results were obtained when the same apoA-I-containing particles were quantified by size exclusion chromatography. Lymph pre-beta apoA-I concentration was low in a subject with familial lecithin:cholesterol acyltransferase deficiency, despite a normal plasma pre-beta apoA-I concentration, but was normal in a subject with familial lipoprotein lipase deficiency. These results suggest that the concentration of small pre-beta HDLs in human tissue fluids is determined only in part by the transfer of pre-beta HDLs across capillary endothelium from plasma. Local production, by remodeling of spheroidal alpha HDLs in tissue fluids, may be equally important. Lipolysis of triglyceride-rich lipoproteins by lipoprotein lipase appears to have little effect.

Do total and high density lipoprotein cholesterol and triglycerides act independently in the prediction of ischemic heart disease? Ten-year follow-up of Caerphilly and Speedwell Cohorts.

Several studies have suggested that men with raised plasma triglycerides (TGs) in combination with adverse levels of other lipids may be at special risk of subsequent ischemic heart disease (IHD). We examined the independent and combined effects of plasma lipids at 10 years of follow-up. We measured fasting TGs, total cholesterol (TC), and high density lipoprotein cholesterol (HDLC) in 4362 men (aged 45 to 63 years) from 2 study populations and reexamined them at intervals during a 10-year follow-up. Major IHD events (death from IHD, clinical myocardial infarction, or ECG-defined myocardial infarction) were recorded. Five hundred thirty-three major IHD events occurred. All 3 lipids were strongly and independently predictive of IHD after 10 years of follow-up. Subjects were then divided into 27 groups (ie, 3(3)) by the tertiles of TGs, TC, and HDLC. The number of events observed in each group was compared with that predicted by a logistic regression model, which included terms for the 3 lipids (without interactions) and potential confounding variables. The incidence of IHD was 22.6% in the group with the lipid risk factor combination with the highest expected risk (high TGs, high TC, and low HDLC) and 4.7% in the group with the lowest expected risk (P<0.01). A comparison of the predicted number of events in the 27 groups with the number of events observed showed that a logistic regression provided an adequate fit without the need to incorporate interactions between lipids in the model. Conclusions are as follows: (1) Serum TGs, TC, and HDLC are independently predictive of IHD at 10 years of follow-up. (2) Combinations of adverse levels of the 3 major lipid risk factors have no greater impact on IHD than that expected from their individual contributions in a logistic regression model. There was no evidence that men with low HDL/raised TGs were at significantly greater risk than that predicted from the independent effects of the 2 lipids considered individually.